TW202014436A - Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods of use thereof - Google Patents

Abstract

Provided herein antibodies, activatable antibodies (AAs), bispecific antibodies, and bispecific activatable antibodies (BAAs). Also provided herein are methods of making and methods of use of these antibodies, AAs, bispecific antibodies, and BAAs.

Description

Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods of using each other

Cross-reference of related applications

In this application, according to 35 USC § 119(e), the US Provisional Application No. 62/666,056 filed on May 2, 2018 and the US Provisional Application No. 62/712,046 filed on July 30, 2018 Interests, each of them is incorporated by reference in this article. Cited sequence listing

According to 37 C.F.R. § 1.821, the sequence list (file name CYTM_060_001TW_SeqList_ST25.txt) of the electronically sent form in computer readable form (ASCII format) via EFS-Web is incorporated herein by reference. The ASCII copy of the sequence table was created on May 1, 2019, and is 224 kilobytes in size. The present invention relates to antibodies, bispecific antibodies, activatable antibodies, and bispecific activatable antibodies, and methods of making and using them.

Antibody-based therapies have proven effective in treating several diseases, but in some cases, toxicity due to extensive target performance has limited their therapeutic effectiveness. In addition, antibody-based therapeutics have exhibited other limitations, such as rapid clearance from circulation after administration.

In the field of small molecule therapeutics, strategies have been developed to provide prodrugs of active chemical entities. These prodrugs are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized into the active compound in the body. These prodrug strategies can provide high selectivity and reduction of adverse reactions of the drug to its desired target.

Therefore, there is a continuing need in the field of antibody-based therapeutics for antibodies that mimic the ideal characteristics of small molecule prodrugs.

Provided herein are antibodies, bispecific antibodies, activatable antibodies, and bispecific activatable antibodies, and methods of making and using each other. These can be used for treatment and diagnosis. The activatable antibodies and bispecific activatable antibodies of the present disclosure can be used to reduce damage to healthy tissue, which is usually caused by the binding of the antibody to healthy tissue and the target of diseased tissue.

In one aspect, provided herein is an activatable antibody (AA), which comprises: (a) at least one scFv comprising a light chain variant region (VL) connected to a heavy chain variant region (VH), wherein the VL is connected to the VH by a linker comprising the amino acid sequence SEQ ID NO: 108 ;and (b) Prodomain, which contains: (i) a masking moiety (MM) coupled to the scFv, wherein the MM reduces or inhibits the binding of the scFV to its target when the AA is in an uncut state; and (ii) a scissible portion (CM) coupled to the scFv, wherein the CM is a substrate polypeptide used as a protease.

In some embodiments, the AB binds to CD3ε.

In another aspect, provided herein is an activatable antibody (AA), which comprises: (a) antibodies or antigen-binding fragments that specifically bind to CD3ε (all collectively referred to as AB); and (b) Prodomain, which contains: (i) a masking portion (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to CD3ε when the AA is in an uncut state, wherein the MM includes the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) A cutable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease.

In another aspect, provided herein is a bispecific activatable antibody (BAA), wherein the BAA specifically binds to two targets when activated, and wherein the BAA is not activated Contains the following structure: a) IgG antibody (AB1) that specifically binds to the first target, where AB1 contains: (i) Two heavy chains (AB1 HC) and two light chains (AB1 LC); and (ii) Two first pre-domains, each of which includes a first masking portion (MM1) connected to the first cuttable portion (CM1) in the direction from N-terminal to C-terminal, wherein each of the first pre-domains The carboxyl end is connected to the amine end of each light chain of AB1, where -The MM1 reduces the binding of the AB1 to its target; and -This CM1 peptide is used as a substrate for the first protease, b) Two scFv (AB2), each of which specifically binds CD3ε, where each AB2 contains: (i) The light chain variation region (VL) connected to the heavy chain variation region (VH), wherein the carboxyl terminal of each AB2 is connected to the amine terminal of each of the AB1 heavy chains, wherein -The VL contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH contains the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and -The VL is connected to the VH by a linker L3, the linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and (ii) Two second pre-domains, each of which includes a second masking portion (MM2) connected to the second cuttable portion (CM2) in the direction from N-terminal to C-terminal, wherein The carboxyl end is connected to the amine end of each AB2, where -The MM2 reduces the binding of AB2 to CD3ε; -The MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and -The CM2 polypeptide is used as a substrate for the second protease.

In another aspect, the provider provided herein is a pharmaceutical composition comprising the AA or BAA and optionally a carrier.

In another aspect, provided herein is a method of treating, reducing symptoms, or delaying progression of a disorder or disease (eg, cancer), which comprises administering a therapeutically effective amount of these AA or BAA or a therapeutically effective amount Pharmaceutical composition (which contains an effective amount of these AA or BAA).

In another aspect, the provided herein is any of the AA, BAA, or pharmaceutical compositions disclosed herein for use as a medicament. In a related aspect, the provided herein is a method for treating AA, BAA, or a pharmaceutical composition of the present disclosure, optionally, wherein the method is used to treat or prevent neoplasms, cancer, Or tumor. In some embodiments, the cancer may be bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, Ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, squamous cell carcinoma, or skin cancer. In some embodiments, the cancer may be related to cells expressing EGFR, for example, tumor cells may include cells expressing EGFR or the tumor microenvironment may include cells expressing EGFR. In some embodiments, provided herein is a method of the disclosed AA, BAA, or pharmaceutical composition for treating a condition or disease (including disease cells expressing EGFR). In some embodiments, provided herein is a method of treatment of the AA, BAA, or pharmaceutical composition of the present disclosure, wherein the treatment includes inhibiting angiogenesis.

In another aspect, the methods provided herein are methods of manufacturing AA and BAA.

Provided herein are antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA).

In some embodiments, provided herein are humanized antibodies that specifically bind to the epsilon chain of CD3 (CD3 epsilon; interchangeably referred to herein as CD3). In some embodiments, provided herein are scFv antibodies that specifically bind to CD3. In some embodiments, provided herein are IgG antibodies that specifically bind to CD3. In some embodiments, the anti-CD3 antibody (eg, anti-CD3 scFv or IgG (such as IgG1) antibody) contains a point mutation in the Fc region, so that the antibody has a reduced effector function.

In some embodiments, provided herein are IgG antibodies that specifically bind to epidermal growth factor receptor (EGFR). In some embodiments, the provider provided herein specifically binds to an IgG1 antibody, wherein the antibody contains a point mutation in the Fc region, so that the antibody has reduced effector function.

In some embodiments, the provided herein is AA, for example, AA that specifically binds to EGFR or CD3. These AA lines are optimized for affinity, effector function, masking, and cutability.

In some embodiments, the provider provided herein is a BAA, for example, bound to a target antigen (eg, tumor antigen, such as the target shown in Table 9) and a second antigen (eg, immune effect on immune effector cells Antigen) BAA. In some embodiments, the immune effector cell line is a white blood cell. In some embodiments, the immune effector cell line is a T cell. In some embodiments, the immune effector cell line is a natural killer (NK) cell. In some embodiments, the immune effector cell line is a macrophage. In some embodiments, the immune effector cells are monocytes, such as myeloid mononuclear cells. In some embodiments, the BAA is immune effector cell-engaging BAA. In some embodiments, the BAA is leukocytic cell-meshing BAA. In some embodiments, the BAA line T cell engagement bispecific (TCB) AA. In some embodiments, the BAA line is a NK cell-meshing BAA. In some embodiments, the BAA is a mating BAA of macrophages. In some embodiments, the BAA is a monocyte-engaging BAA, such as bone marrow-like monocyte-engaging BAA. In some embodiments, BAA binds EGFR and CD3. These BAA lines are optimized for affinity, effector function, masking, and cutability.

Also provided herein are the methods of making and using these antibodies, AA and BAA. AA, including its general manufacturing method and identification masking part (MM) and cutable part (CM), is described in International Publication No. WO 2009/025846 of Daugherty et al., published on February 26, 2009; and Stagliano WO 2010/081173 et al., published on July 15, 2010, both of which are incorporated by reference. BAA, including its general manufacturing method and identification masking part (MM) and cutable part (CM), are described in International Publication No. WO2015/013671 of Lowman et al., published on January 29, 2015; and Irving etc. Renzhi WO2016/014974, published on January 28, 2016, both of which are fully incorporated by reference. Also incorporated by reference are International Publication No. WO2016/014974 of Irving et al., published on January 28, 2016, and International Publication No. WO2016/118629 of Moore et al., published on July 28, 2016. Provide AA, general manufacturing, MM, and CM.

As used herein, unless otherwise stated, the term "antibody" includes antibodies or antigen-binding fragments that specifically bind to its target, and are monoclonal antibodies, domain antibodies, single chain, Fab fragments, F(ab')2 fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, the anti-system IgG antibody. In some embodiments, the anti-system IgG1 antibody. In some embodiments, the anti-system IgG4 antibody. In some embodiments, the anti-system scFv antibody. In some embodiments, the antibody or immunologically active fragment that binds to its target is a mouse, chimeric, humanized, or fully human monoclonal antibody. 1. CD3 antibody

Provided herein are antibodies or antigen-binding fragments (AB) that specifically bind to the epsilon chain of CD3 (CD3 epsilon; referred to herein as CD3). Provided herein are activatable antibodies (AA) and bispecific activatable antibodies (BAA), which include these CD3 AB.

Table 1 provides exemplary amino acid sequences (variant domains) of the CD3 binding antibodies disclosed in Table 1. (The underlined ones are the predicted CDR sequences). As provided below, the L3 line linker connects the light chain and heavy chain variant domains in an exemplary CD3 binding antibody.

Exemplary scFv linkers (referred to herein as "L3", which connects VL and VH) are provided in Table 1-1.

Exemplary CDR sequences for CD3 binding antibodies are provided in Table 2.

As provided herein, the CD3 antibody comprises at least one, at least two, at least three, at least four, or at least five of the CDR sequences provided in Table 2. In some embodiments, the CD3 antibody comprises the six CDR sequences provided in Table 2.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 2.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 3.

In some embodiments, the CD3 antibody comprises the light chain variant domain shown in SEQ ID NO: 1.

In some embodiments, the CD3 antibody comprises the light chain variant domain shown in SEQ ID NO: 4.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 2 and the light chain variant domain shown in SEQ ID NO: 1.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 3 and the light chain variant domain shown in SEQ ID NO: 1.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 3 and the light chain variant domain shown in SEQ ID NO: 4.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 2 and the light chain variant domain shown in SEQ ID NO: 4.

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 2 or SEQ ID NO: 3 or the light chain variant domain shown in SEQ ID NO: 1 or SEQ ID NO: 4. .

In some embodiments, the CD3 antibody comprises the heavy chain variant domain shown in SEQ ID NO: 2 or SEQ ID NO: 3 and the light chain variant domain shown in SEQ ID NO: 1 or SEQ ID NO: 4. .

In some embodiments, the antibody comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences to the above heavy chain variant domain, and/or comprises sequences 70%, 80%, 90% , 70%, 80%, 90%, 95%, 99%, or 100% similar to the light chain variation domain as described above, and specifically binds to CD3. In some embodiments, the heavy chain variant domain is combined with the light chain variant domain as described above. In some embodiments, the antibody comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences to the above heavy chain variant domain, and/or comprises sequences 70%, 80%, 90% The sequence similar to 70%, 80%, 90%, 95%, 99%, or 100% of the light chain variation domain as described above includes the six CDR sequences provided in Table 2 and specifically binds to CD3.

In some embodiments, the CD3 anti-system scFv antibody. In some embodiments, the variation domain includes the following structure from N-terminal to C-terminal: LV-HV. In some embodiments, the variation domain comprises the following structure from N-terminus to C-terminus: HV-LV.

In some embodiments, the CD3 anti-system scFv antibody comprises a light chain variant region (VL) linked to a heavy chain variant region (VH), wherein the VL is composed of an amino acid sequence of SEQ ID NO: 98 The linker is connected to the VH. Exemplary sequences with this linker are provided in Table 1.

In some embodiments, the CD3 anti-system scFv antibody comprises a light chain variant region (VL) linked to a heavy chain variant region (VH), wherein the VL is composed of an amino acid sequence of SEQ ID NO: 108 The linker is connected to the VH. Exemplary sequences with this linker are provided in Table 1.

In an exemplary embodiment, provided herein is an antibody (AB) that specifically binds to CD3, wherein the anti-system IgG1 antibody or scFv linked to the Fc domain, wherein the antibody comprises an Fc region, which is contained in Amino acid substitution of at least one of amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat) makes the antibody have a reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions of L234F, L235E, and P331S. In some embodiments, the antibody comprises an Fc region, which contains an amino acid substitution in N297. In some embodiments, the Fc region contains the N297Q mutation. In some embodiments, the antibody comprises amino acid substitutions of L234F, L235E, P331S, and N297Q. In some embodiments, the heavy chain variant domain of the antibody with reduced effector function comprises any one of SEQ ID NO: 2 or SEQ ID NO: 3 or wherein the AB light chain variant domain comprises SEQ ID NO : 1 or any of SEQ ID NO: 4. 2. The activatable CD3 antibody

In some embodiments, any of the CD3 antibodies provided herein is in an activatable antibody (AA) format.

As provided generally herein, the AA of the present disclosure includes the MM-CM construct, also referred to herein as the pre-domain. Therefore, as used herein, the term "prodomain" refers to a polypeptide that includes a masking portion (MM) and a truncable portion (CM). In some embodiments, MM and CM are separated by a linker, referred to herein as L1. In some embodiments, the pre-domain includes a linker at the carboxyl end of CM; this linker, in Called L2 herein, the CM of the pre-domain is connected to AB. In some embodiments, the pre-domain comprises a linker between MM and CM and a linker after CM. In some implementations, MM and CM are not separated by linkers. In some embodiments, the pre-domain comprises one of the following formulas (wherein the following formulas represent the amino acid sequence in the N-C-terminal direction or the C-N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In an exemplary embodiment, the prodomain comprises EGFR MM and CM that can be cut by matriptase or MMP; or CD3ε MM and CM that can be cut by matriptase or MMP. In some embodiments, the prodomain comprises EGFR MM and CM that can be cleaved by interstitial proteases and MMP. In some embodiments, the prodomain comprises CD3ε MM and CM that can be cleaved by interstitial proteases and MMP. Provided herein are activatable antibodies (AA), and contain the pro-domain. Also provided herein are the nucleotides encoding the ellipse domain before the disclosure.

Therefore, in some embodiments, provided herein is CD3 AA, which comprises: (a) an antibody or antigen-binding fragment (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein the antibody Contains the heavy chain domain shown in SEQ ID NO: 2 or SEQ ID NO: 3 or the light chain domain shown in SEQ ID NO: 1 or SEQ ID NO: 4; and (b) the pre-domain, wherein the The pre-domain includes (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to CD3ε when the AA is in an uncut state; and (ii) A cutable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease.

In some embodiments, provided herein is CD3 AA, which comprises: (a) an antibody or antigen-binding fragment (AB) that specifically binds to CD3ε; and (b) a pre-domain, wherein the pre The domain contains: (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to CD3ε when the AA is in an uncut state, wherein the MM contains an amine Base acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) a cleavable portion (CM) coupled to the AB, wherein the CM is used as a protease receptor Qualitative polypeptide.

In some embodiments, provided herein is CD3 AA, which comprises: (a) at least one scFv, which comprises a light chain variant region (VL) connected to a heavy chain variant region (VH), wherein the VL Is connected to the VH by a linker comprising the amino acid sequence SEQ ID NO: 108 or SEQ ID NO: 98; and (b) a prodomain, which includes: (i) coupled to the scFv A masking moiety (MM), wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the scFV to its target; and (ii) the scFv can be coupled to the cutable Part (CM), wherein the CM is a substrate polypeptide used as a protease.

Therefore, in some embodiments, provided herein is CD3 AA, which when unactivated, contains the following structure: (a) when activated, specifically binds to the ε chain of CD3 (CD3ε) Antibody or antigen-binding fragment (AB), wherein the antibody comprises 70%, 80%, 90%, 95%, 99%, or 100% similar to the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 The heavy chain domain may include a light chain domain that is 70%, 80%, 90%, 95%, 99%, or 100% similar to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; and (b) before Domain, wherein the pre-domain contains (i) a masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the AB and CD3ε binding; and (ii) a cleavable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease. The AB may include at least one of the CDR sequences provided in Table 2. The AB may contain the six CDR sequences provided in Table 2.

In some embodiments, provided herein is CD3ε AA, which specifically binds to a target when activated, and wherein the AA contains the following structure when not activated: (a) an antibody that specifically binds to CD3ε Or its antigen-binding fragment (AB); and (b) predomain (prodomain), which includes: (i) a masking moiety (MM) coupled to the AB, where the AA is in an uncut state , The MM reduces or inhibits the binding of AB to CD3ε, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) is coupled to the AB Cutable part (CM), wherein the CM is used as a substrate for proteases.

In some embodiments, the AA provided herein specifically binds to the target when activated, and wherein the AA contains the following structure when not activated: (a) an antibody that specifically binds to CD3ε or Its antigen-binding fragment (AB); and (b) predomain (prodomain), which includes: (i) a masking moiety (MM) coupled to the AB, where the AA is in an uncut state , The MM reduces or inhibits the binding of the AB to CD3ε, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) may be coupled to the AB The cut part (CM), wherein the CM is used as a substrate for proteases.

In some embodiments, the AB of CD3 AA is any of the CD3 antibodies described in the previous section.

In some embodiments, the AB of CD3 AA comprises the heavy chain variant domain shown in SEQ ID NO: 2.

In some embodiments, the AB of CD3 AA comprises the heavy chain variant domain shown in SEQ ID NO: 3.

In some embodiments, the AB of CD3 AA comprises the light chain variant domain shown in SEQ ID NO: 1.

In some embodiments, the AB of CD3 AA comprises the light chain variant domain shown in SEQ ID NO: 4.

In some embodiments, the AB of CD3 AA comprises the heavy chain variant domain shown in SEQ ID NO: 2 and the light chain domain shown in SEQ ID NO: 1.

In some embodiments, the AB of CD3 AA includes the heavy chain variant domain shown in SEQ ID NO: 3 and the light chain domain shown in SEQ ID NO: 1.

In some embodiments, the AB of CD3 AA includes the heavy chain variant domain shown in SEQ ID NO: 3 and the light chain domain shown in SEQ ID NO: 4.

In some embodiments, the AB is an scFv that includes a light chain variant region (VL) linked to a heavy chain variant region (VH), wherein the VL is linked by an amino acid sequence including SEQ ID NO: 108 The child is connected to the VH. Exemplary sequences with this linker are provided in Table 1.

In some embodiments, the AB is an scFv that includes a light chain variant region (VL) linked to a heavy chain variant region (VH), wherein the VL is linked by an amino acid sequence SEQ ID NO: 98 The child is connected to the VH. Exemplary sequences with this linker are provided in Table 1.

In some embodiments, the MM of CD3 AA comprises any of the sequences shown in Table 3.

Exemplary CD3 masking parts (MM) of the present disclosure are provided in Table 3.

In some embodiments, the MM of the CD3 AA comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the MM of the CD3 AA is the sequence shown in SEQ ID NO: 10. In some embodiments, the MM of the CD3 AA is the sequence shown in SEQ ID NO: 11. In some embodiments, the MM of the CD3 AA comprises the sequence shown in SEQ ID NO: 105. In some embodiments, the MM of the CD3 AA comprises the sequence shown in SEQ ID NO: 106. In some embodiments, the MM of the CD3 AA comprises the sequence shown in SEQ ID NO: 107.

In some embodiments, the CM of CD3 AA includes any of the sequences shown in Table 4. Exemplary cut-off parts (CM) of this disclosure are provided in Table 4. In some embodiments, the truncable portion of CD3 AA comprises any amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

3. 具有 Fc 突變之抗體 In some embodiments, the CM of AA disclosed herein includes any of the sequences shown in Table 4-1. In some embodiments, the CM of AA of the present disclosure includes any amino acid sequence selected from the group consisting of SEQ ID NO: 18-56.

3. Antibodies with Fc mutations

Provided herein are IgG1 antibodies with Fc mutations or antibody fragments containing an antigen binding domain (eg, scFv, Fab, F(ab')2) linked to an Fc domain, wherein the Fc exhibits reduced effector function (Referred to herein as Fc variants).

Antibodies containing these Fc mutations will result in reduced effector function while maintaining target binding affinity. Therefore, provided herein are antibodies that bind to the target of interest, wherein the anti-system IgG1 antibody or antibody fragment linked to Fc, wherein the Fc region includes amino acid positions L234, L235, and P331 ( Substitution by at least one of the amino acids in the EU index number shown in Kabat) makes the antibody have a reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, there is an additional mutation at N297. In some embodiments, the amino acid substitution is N297Q or N297A.

In some embodiments, the Fc is selected from the Fc sequences presented in Table 4-2.

Provided herein are antibodies, AA, bispecific antibodies, and BAA containing these Fc mutations.

In some embodiments, the Fc domains of these antibodies, AA, bispecific antibodies, and BAA comprise any of the sequences shown in Table 4-2: SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122.

In some embodiments, these Fc variant-containing AA and BAA can bind immune effector cells. In some embodiments, they can bind targets that are selectively located on immune effector cells. In some embodiments, they can bind CD3. In some embodiments, they can bind any of the targets listed in Table 9. In some embodiments, they can bind EGFR.

Therefore, in some embodiments, provided herein is an activatable antibody (AA), which comprises: an antibody (AB) that specifically binds to a target, wherein the anti-system IgG1 antibody, and wherein the antibody The Fc region includes amino acid substitutions of at least one of amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat) so that the AA has a reduced effector function; and the pre-domain, The pre-domain contains: (i) a masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB to the target; and (ii) A cutable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease.

Therefore, in some embodiments, provided herein is an activatable antibody (AA), which comprises: an antibody (AB) that specifically binds to a target, wherein the anti-system IgG1 antibody, and wherein the antibody The Fc region includes amino acid substitutions of at least one of amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat) so that the AA has a reduced effector function; and the pre-domain, The pre-domain contains: (i) A masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB to the target, wherein the MM contains an amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) A cutable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease.

In some embodiments, the Fc region includes at least amino acid positions L234, L235, N297, and P331 (eg, L234F, L235E, P331S, and/or N297A or N297Q) (numbered by the EU index shown in Kabat) The amino acid substitution makes the AA have a reduced effector function. In some embodiments, the target is selected from the group consisting of the targets presented in Table 9.

In some embodiments, provided herein is a bispecific activatable antibody (BAA), wherein the BAA specifically binds to two targets (or binds to two targets on the same target when activated) Different epitopes), and wherein the BAA contains the following structure when not activated: a) IgG antibody (AB1) that specifically binds to the first target, wherein the AB1 contains: (i) two heavy chains (AB1 HC ) And two light chains (AB1 LC); and (ii) two first pre-domains, each of which includes a first masking portion (CM1) connected to the first cleavable portion (CM1) in the direction from N-terminal to C-terminal ( MM1), wherein the carboxy terminus of each first pro-domain is connected to the amine terminus of each light chain of the AB1, wherein the MM reduces or inhibits the binding of the AB1 to its target; and the CM1 is a polypeptide used to As the substrate of the first protease, b) two scFv (AB2), each of which specifically binds CD3ε, where each AB2 contains: (i) a light chain variant region (VL) connected to a heavy chain variant region (VH), The carboxyl end of each AB2 is connected to the amine end of each of the AB1 heavy chains, wherein the VL contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH contains SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3; and the VL is connected to the VH by a linker (L3), the linker comprises a group selected from SEQ ID NO: 98 and SEQ ID NO : The amino acid sequence of the group consisting of 108; and (ii) two second pre-domains, each of which includes a second connected to the second cleavable portion (CM2) in the N-terminal to C-terminal direction The masking part (MM2), wherein the carboxyl end of each second pre-domain is connected to the amine end of each AB2, wherein the MM2 reduces or inhibits the binding of AB2 to CD3ε; the MM2 includes a group selected from the group consisting of Amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and the CM2 polypeptide, which is used as a substrate for the second protease. 4. EGFR antibody

The providers provided herein are antibodies or antigen-binding fragments (AB) that specifically bind to EGFR. Exemplary CDR sequences for EGFR-binding antibodies are provided in Table 5.

Provided herein are EGFR antibodies, bispecific antibodies that target EGFR, AA that can bind EGFR when activated, and BAA that can bind EGFR when activated. In some embodiments, the EGFR antibody comprises the CDRs of Table 5.

In some embodiments, such as in the form of BAA, the providers provided herein are IgG antibodies that specifically bind to epidermal growth factor receptor (EGFR) and confer reduced effector function. Antibodies containing Fc mutations will result in reduced effector function while maintaining EGFR binding affinity. Therefore, provided herein is an antibody that binds to EGFR, wherein the anti-system IgG1 antibody, wherein the antibody comprises an Fc region, which comprises amino acid positions L234, L235, and P331 (by the EU shown in Kabat Index number) at least one of the amino acid substitutions makes the antibody have a reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S.

In some embodiments, the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331.

In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331.

In some embodiments, the antibody comprises amino acid substitutions of L234F, L235E, and P331S.

In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297, plus an amino acid substitution at at least one of amino acid positions L234, L235, and/or P331. In some embodiments, the Fc region contains the N297Q mutation. In some embodiments, the Fc region contains the N297A mutation.

In some embodiments, the antibody comprises L234F, L235E, P331S, and N297Q substitutions. In some embodiments, the antibody comprises L234F, L235E, P331S, and N297A substitutions.

Exemplary CDR sequences for EGFR-binding antibodies are provided in Table 5 and shown in Kabat format.

Exemplary amino acid sequences of EGFR-binding antibodies are provided in Table 6. (VL and VH represent the variant light chain and variant heavy chain, respectively; LC and HC represent the light chain and heavy chain, respectively).

In some embodiments, the EGFR antibody comprises any of the sequences provided in Table 6.

In some embodiments, the heavy chain of the EGFR antibody comprises any of the sequences shown in Table 6: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76.

The symbol Fcmt3 contains three key mutations, wherein the Fc region of the EGFR antibody heavy chain contains the following three point mutations: L234F, L235E, and P331S. Therefore, in some embodiments, the EGFR antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: C225v5Fcmt3 HC. In some embodiments, the Fc region of the EGFR antibody heavy chain contains a fourth point mutation N297Q. The symbol Fcmt4 contains the Fcmt3 three-point mutation and the fourth point mutation N297Q. Therefore, in these embodiments, the EGFR antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 76.

5. 可活化之 EGFR 抗體 In some embodiments, the AB comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences to the heavy chain variation domain shown in SEQ ID NO: 64, and/or comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences of the light chain variation domain shown in SEQ ID NO: 63, and specifically bind to EGFR. In some embodiments, the AB comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences to the heavy chain variation domain shown in SEQ ID NO: 64, and/or comprises 70%, 80%, 90%, 95%, 99%, or 100% similar sequences of the light chain variation domain shown in SEQ ID NO: 63, including the six CDR sequences provided in Table 5, and specifically binding To EGFR. In some embodiments, AB comprises the Fc region as shown above or in Table 6.

The EGFR antibody can activate the

In some embodiments, any of the EGFR anti-systems provided herein are in AA format (EGFR AA). Therefore, provided herein is AA, which comprises an antibody or antigen-binding fragment (AB) that specifically binds to EGFR. Exemplary CDR sequences for EGFR-binding antibodies are provided in Table 5.

In some embodiments, the AA comprises: (a) any antibody or antigen-binding fragment (AB) that specifically binds to epidermal growth factor receptor (EGFR); and (b) a prodomain, wherein The pre-domain includes: (i) a masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB to EGFR, and wherein the MM contains an amino acid sequence selected from the group consisting of the sequences presented in Table 7; and (ii) a cleavable portion (CM) coupled to the AB, wherein the CM is used as a protease Peptide.

Exemplary EGFR masking parts (MM) of the present disclosure are provided in Table 7 and Table 8.

In some embodiments, the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the CM of the EGFR AA includes an amino acid sequence selected from the group consisting of the sequences presented in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, provided herein is an activatable antibody (AA), which comprises: (a) an antibody that specifically binds to epidermal growth factor receptor (EGFR), wherein the anti-system IgG1 antibody, And wherein the Fc region of the antibody includes amino acid substitutions of at least one of amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat), so that the AA has a reduced effector function; (b) A masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB to EGFR; and (c) and The cutable portion (CM) of the AB coupling, wherein the CM is a substrate polypeptide used as a protease. The EGFR IgG1 antibody may be any IgG1 antibody described in the previous section. In some embodiments, the MM includes an amino acid sequence selected from the group consisting of the sequences presented in Table 7.

In an exemplary embodiment, provided herein is an activatable antibody (AA), which includes: (a) an antibody (AB) that specifically binds to epidermal growth factor receptor (EGFR), wherein the AB is an IgG1 antibody, and the Fc region of the AB includes amino acid substitutions of at least one of amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat), so that the AA has a decrease Effector function; (b) a masking moiety (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB to EGFR; And (c) a cutable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the AB includes amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the AB includes amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the AB includes amino acid substitutions of L234F, L235E, and P331S. In some embodiments, the AB includes an Fc region, which includes an amino acid substitution in N297. In some embodiments, the Fc region contains the N297Q mutation. In some embodiments, the AB includes amino acid substitutions of L234F, L235E, P331S, and N297Q. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of the sequences presented in Table 7 or Table 8. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CM includes an amino acid sequence selected from the group consisting of the sequences presented in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the AA is part of the BAA. 6. Bispecific activatable antibody (BAA)

Provided herein is BAA (Bispecific AA, BAA), where the bispecific AA specifically binds two targets when activated (eg, binds two different targets, or binds to the same target Two different epitopes) and can contain the exemplary structure provided in FIG. 17.

In some embodiments, the first target is selected from the group consisting of the targets presented in Table 9, and the second target is selected from the group consisting of the targets presented in Table 9.

As provided generally herein, and as described in the AA section above, the BAA of the present disclosure includes a MM-CM construct, also referred to herein as a pre-domain. Therefore, as used herein, the term "prodomain" refers to a polypeptide that includes a masking portion (MM) and a truncable portion (CM). In some embodiments, MM and CM are separated by a linker (referred to herein as L1). In some embodiments, the pro-domain comprises a linker at the carboxy-terminus of the CM; this linker, referred to herein as L2, connects the CM of the pro-domain to AB. In some embodiments, the pre-domain comprises a linker between MM and CM and a linker after CM. In some implementations, MM and CM are not separated by linkers. In some embodiments, the pre-domain comprises one of the following formulas (wherein the following formulas represent the amino acid sequence in the N-C-terminal direction or the C-N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In an exemplary embodiment, the prodomain comprises EGFR MM and CM that can be cut by matriptase or MMP; or CD3ε MM and CM that can be cut by matriptase or MMP. In some embodiments, the prodomain comprises EGFR MM and CM that can be cleaved by interstitial proteases and MMP. In some embodiments, the prodomain comprises CD3ε MM and CM that can be cleaved by interstitial proteases and MMP. Provided herein is a bispecific activatable antibody (BAA) and contains a prodomain. Also provided herein are the nucleotides encoding the ellipse domain before the disclosure.

In some embodiments, the provider provided herein is a BAA, where the BAA specifically binds to two targets (eg, two different targets; or two different tables on the same target when activated) Position), and the BAA contains the following structure when not activated: a) IgG antibody (AB1) that specifically binds to the first target, where AB1 contains: i. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. Two first pre-domains, each of which contains a first masking portion (MM1) connected to the first cleavable portion (CM1) from the N-terminal to the C-terminal direction, wherein the carboxyl group of each first pre-domain The end is connected to the amine end of each light chain of AB1, where The MM1 reduces or inhibits the binding of the AB1 to its target; and This CM1 polypeptide is used as a substrate for the first protease, b) Two scFv (AB2), each of which specifically binds CD3ε, where each AB2 contains: i. The light chain variation region (VL) connected to the heavy chain variation region (VH), wherein the carboxyl end of each AB2 is connected to the amine end of each of the AB1 heavy chains, wherein the VL contains SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 4, and the VH includes the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and the VL is through the linker (L3) Linked to the VH, the linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and ii. Two second pre-domains, each of which contains a second masking portion (MM2) connected to the second cleavable portion (CM2) in the direction from N-terminal to C-terminal, wherein the carboxyl group of each second pre-domain The end is connected to the amine end of each AB2, where The MM2 reduces the binding of AB2 to CD3ε; The MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and The CM2 is a polypeptide used as a substrate for the second protease.

In some embodiments, the VL is connected to the VH via a linker (L3) comprising the amino acid sequence SEQ ID NO: 98.

In some embodiments, the VL is connected to the VH through a linker (L3) comprising the amino acid sequence SEQ ID NO: 108.

In some embodiments, the MM2 comprises the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the MM2 comprises the amino acid sequence of SEQ ID NO: 105.

In some embodiments, the MM2 comprises the amino acid sequence of SEQ ID NO: 106.

In some embodiments, the MM2 comprises the amino acid sequence of SEQ ID NO: 107.

In some embodiments, the AB1 binds to a tumor target.

In some embodiments, the AB1 binds EGFR. As disclosed herein, EGFR binding AB1 can be any EGFR binding antibody or fragment thereof.

In some embodiments, as disclosed herein, AB2 can be any CD3 binding antibody or fragment thereof.

In some embodiments, the MM1 comprises an amino acid sequence selected from the group consisting of the sequences presented in Table 7 or Table 8.

In some embodiments, the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 85.

In some embodiments, the MM1 comprises the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the MM1 comprises the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the CM1 comprises the amino acid sequence of any of the CMs listed in Table 4 or Table 4-1.

In some embodiments, the CM2 comprises the amino acid sequence of any of the CMs listed in Table 4 or Table 4-1.

In some embodiments, the CM1 or CM2 comprises the amino acid sequence of any of the CMs listed in Table 4 or Table 4-1.

In some embodiments, the CM1 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.

In some embodiments, the CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.

In some embodiments, the CM1 or CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.

In some embodiments, the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.

In some embodiments, the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.

In some embodiments, the AB1 includes an Fc region that includes an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331 (numbered by the EU index shown in Kabat), such that The BAA has a reduced effector function.

In some embodiments, the AB1 includes amino acid substitutions in at least two of amino acid positions L234, L235, and P331.

In some embodiments, the AB1 includes amino acid substitutions at amino acid positions L234, L235, and P331.

In some embodiments, the AB1 includes amino acid substitutions of L234F, L235E, and P331S.

In some embodiments, the AB1 comprises an Fc region, which includes an amino acid substitution in N297.

In some embodiments, the AB1 includes amino acid substitutions of L234F, L235E, P331S, and N297Q.

In some embodiments, the heavy chain of AB1 comprises the amino acid sequence shown in Table 6 selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76.

In some embodiments, the BAA is CI138, which contains the layouts and sequences provided in Tables 11 and 1. In some embodiments, the BAA is CI138, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 155 or SEQ ID NO: 124, and the light chain sequence comprises a The amino acid sequence of the group consisting of SEQ ID NO: 132 or SEQ ID NO: 140.

In some embodiments, the BAA is CI139, which contains the layouts and sequences provided in Tables 11 and 1. . In some embodiments, the BAA is CI139, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 157 or SEQ ID NO: 126, and the light chain sequence comprises a The amino acid sequence of the group consisting of SEQ ID NO: 132 or SEQ ID NO: 140.

In some embodiments, the BAA is CI158, which contains the layouts and sequences provided in Tables 11 and 1. In some embodiments, the BAA is CI158, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 161 or SEQ ID NO: 128, and the light chain sequence comprises a The amino acid sequence of the group consisting of SEQ ID NO: 132 or SEQ ID NO: 140.

In some embodiments, the BAA is CI140, which includes the layouts and sequences provided in Tables 11 and 1. In some embodiments, the BAA is CI140, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 159 or SEQ ID NO: 25, and the light chain sequence comprises a The amino acid sequence of the group consisting of SEQ ID NO: 132 or SEQ ID NO: 140.

In some embodiments, the BAA provided herein includes: c) IgG antibody (AB1) that specifically binds to the first target, where AB1 contains: i. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. The first masking portion (MM1) connected to the first cleavable portion (CM1) to form a pre-domain (herein interchangeably with MM1-CM1 construct), wherein the carboxy-terminus of the pre-domain It is connected to each amine end of each light chain of AB1, where 1. The MM1 inhibits the binding of the AB1 to its target; and 2. The CM1 peptide is used as a substrate for the first protease, d) Two scFv (AB2), each of which specifically binds to the second target, where each AB2 contains: i. The heavy chain variation region connected to the light chain variation region, wherein the carboxyl end of each AB2 is connected to the amine end of each AB1 heavy chain; and ii. a second masking portion (MM2) connected to the second cleavable portion (CM2) to form a second pre-domain, wherein the carboxyl end of the second pre-domain is connected to the amine end of each AB2, wherein The MM2 inhibits the binding of the AB2 to its target; and The CM2 is a polypeptide, which is used as a substrate for the second protease, And the BAA has the following characteristics: i. The MM2 contains the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106, SEQ ID NO: 107; ii. MM1 contains an amino acid sequence selected from the group consisting of the sequences presented in Table 7; iii. AB2 contains the heavy chain variant domain shown in SEQ ID NO: 2 or SEQ ID NO: 3 or the light chain variant domain shown in SEQ ID NO: 1 or SEQ ID NO: 4; iv. AB1 includes an Fc region that includes amino acid substitutions at amino acid positions L234, L235, N297, and P331 or at least one of L234, L235, and P331 (numbered by the EU index shown in Kabat), such that The BAA has a reduced effector function.

In some embodiments, the BAA provided herein comprises: a) an IgG antibody (AB1) that specifically binds to the first target, wherein the AB1 comprises: a. two heavy chains (AB1 HC) and two light chains Chain (AB1 LC); and b. a first masking portion (MM1) connected to the first cleavable portion (CM1) to form a pre-domain, wherein the carboxyl end of the pre-domain is connected to each light chain of the AB1 The amine ends of the amine groups are connected, of which 1. The MM1 inhibits the binding of AB1 to its target; and 2. The CM1 is a polypeptide used as a substrate for the first protease, b) two scFv (AB2), which Each specifically binds to the second target, where each AB2 includes: a. a heavy chain variation region connected to the light chain variation region, wherein the carboxyl end of each AB2 is connected to the amine end of each AB1 heavy chain; And b. a second masking part (MM2) connected to the second cleavable part (CM2) to form a pre-domain, wherein the carboxyl end of the pre-domain is connected to the amine end of each AB2, wherein 1. MM2 inhibits the binding of the AB2 to its target; and 2. The CM2 polypeptide, which is used as a substrate for the second protease, and wherein the AB1 contains an Fc region, which is contained at amino acid positions L234, L235, N297 , And amino acid substitution of at least one of P331 (EU index number shown in Kabat), so that the BAA has a reduced effector function. In some embodiments, the Fc region includes amino acid substitutions of at least two of amino acid positions L234, L235, N297, and P331 (numbered by the EU index shown in Kabat), so that the BAA has a reduction Effector function.

In some embodiments, the Fc region includes amino acid substitutions at least at amino acid positions L234, L235, and P331 (numbered by the EU index shown in Kabat), so that the BAA has a reduced effector function. In some embodiments, the first target is selected from the group consisting of the targets presented in Table 9, and the second target is selected from the group consisting of the targets presented in Table 9.

In some embodiments, AB1 binds to a target antigen, such as a tumor antigen, and AB2 binds to an immune effector target.

In some embodiments, AB2 binds to a target antigen, such as a tumor antigen, and the AB1 binds to an immune effector target.

In some embodiments, AB1 binds EGFR and AB2 binds CD3. In some embodiments, the AB1 is an EGFR binding antibody or fragment thereof as disclosed herein, and AB2 is a CD3 binding antibody or fragment thereof as disclosed herein.

In some embodiments, the MM1 comprises an amino acid sequence selected from SEQ ID NO: 78-87. In some embodiments, the MM1 comprises SEQ ID NO: 78.

In some embodiments, the MM2 comprises an amino acid sequence selected from SEQ ID NOs: 12, 106, 107, and 105. In some embodiments, the MM2 comprises the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the bispecific AA is CI106, CI138, CI139, CI158, or CI140, as provided in Table 11 in Example 1.

In an exemplary embodiment, AB1 comprises the amino acid sequence of C225v5Fcmt3 HC or C225v5Fcmt4 HC.

In some embodiments, the first and second proteases are the same protease. In some embodiments, the first and second proteases are different proteases. In some embodiments, CM1 and CM2 comprise the same amino acid sequence. In some embodiments, CM1 and CM2 comprise different amino acid sequences. In some embodiments, CM1 and CM2 comprise different amino acid sequences, which can be cleaved by the same protease or proteases. In some embodiments, CM1 and CM2 can be cleaved by more than one protease. In some embodiments, CM1 and/or CM2 can be cleaved by serine proteases. In some embodiments, CM1 and/or CM2 can be cleaved by matrix metalloproteinase (MMP). In some embodiments, CM1 and/or CM2 can be cut by serine protease and MMP.

Exemplary BAAs of the present disclosure include, for example, those shown in the embodiments provided herein and variants thereof.

In some non-limiting embodiments, at least one of the ABs in the BAA is specific for CD3, and at least one other AB is a binding partner of any of the targets listed in Table 9.

In an exemplary embodiment, AB in BAA is specific for CD3 and AB1 is a binding partner for any of the targets listed in Table 9.

In some embodiments, the unmasked EGFR-CD3 bispecific antibody exhibits EGFR-dependent tumor cell killing, while the double-masked EGFR-CD3 BAA reduces target-dependent cytotoxicity by more than 100,000-fold. In established tumor models where tumor-resident proteases are expected to be active, BAA was shown to effectively induce tumor regression. In non-human primates, the maximum tolerable dose (MTD) of EGFR-CD3 BAA is more than 60 times larger than the MTD of unmasked bispecific antibodies, and the tolerable exposure (AUC) is more than 10,000 times larger . Despite a 60-fold dose difference in MTD, the transient increase in serum interleukins and AST/ALT observed in non-human primates treated with BAA is still lower than that induced by bispecific antibodies. By positioning the activity in the tumor microenvironment, BAA has the potential to expand the clinical opportunities for T cell engagement bispecific therapy that is limited by target toxicity, especially in solid tumors. In addition, EGFR-CD3 BAA has the potential to treat EGFR-expressing tumors that respond poorly to existing EGFR-directed therapies. 7. Cutable part (CM)

When masked and not activated, both monospecific AA and BAA of the present disclosure include at least one CM.

In some embodiments, the truncable portion (CM) of AA or BAA includes an amino acid sequence that can serve as a substrate for at least one protease, usually an extracellular protease. In the case of AA or BAA, CM can be selected based on proteases co-located in the tissue with at least one AB of BAA or the desired target of AB of AA. CM can be used as a substrate for various proteases, such as serine protease and a second different protease (eg, MMP). In some embodiments, CM can serve as a substrate for more than one serine protease (eg, interstitial protease and uPA). In some embodiments, CM can be used as a substrate for more than one MMP (eg, MMP9 and MMP14).

A variety of different conditions are known, where the target of interest is co-located with the protease, and the substrate of the protease is known in the art. In the case of cancer, the target tissue may be cancer tissue, especially cancer tissue of solid tumor. The literature reports an increase in protease levels in many cancers, such as liquid tumors or solid tumors. See, for example, La Rocca et al. (2004) British J. of Cancer 90(7): 1414-1421. Non-limiting examples of diseases include: all types of cancer (such as but not limited to breast cancer, lung cancer, colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, endometrial cancer, renal cancer, sarcoma, skin cancer, cancer Cervical cancer, liver cancer, bladder cancer, bile duct cancer, prostate cancer, melanoma, head and neck cancer (such as head and neck squamous cell carcinoma, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, local deficiency Blood, etc. For example, indications include leukemia, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic disease including multiple myeloma, and lung cancer, colorectal cancer, prostate cancer, pancreas Solid tumors of cancer and breast cancer (including triple negative breast cancer). For example, indications include bone disease or cancer metastasis, regardless of the origin of the primary tumor; breast cancer, which includes but is not limited to ER/PR+ breast cancer, Her2+ breast cancer, triple negative Breast cancer; Colorectal cancer; Endometrial cancer; Gastric cancer; Glioblastoma; Head and neck cancer, such as head and neck squamous cell carcinoma; Esophageal cancer; Lung cancer, such as, as a non-limiting example, non-small cell lung cancer; Multiple myeloma Ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as a non-limiting example, renal cell carcinoma; and/or skin cancer, such as, as a non-limiting example, squamous cell carcinoma, basal cell carcinoma , Or melanoma

The rate at which CM is specifically cleaved by enzymes is about .001-1500 x 10 ⁴ M ^-1 S ^-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, ^1, 2.5, 5, 7.5, 10, 15, ²⁰ , 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 ⁴ M ^-1 S ^-1 .

In order to be specifically cut by the enzyme, the contact between the enzyme and the CM is performed. When AA or BAA contains at least a first AB coupled to MM and CM, for example, AA or BAA contains AB coupled to MM via CM, and CM can be truncated in the presence of target and sufficient enzyme activity. Sufficient enzyme activity may refer to the ability of the enzyme to contact the CM and achieve a cut. It is easy to foresee that the enzyme can be near the CM, but it cannot be cut due to protein modification of other cytokines or enzymes.

The exemplary CM of this disclosure is provided in Table 4 above. In some embodiments, the CM has a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, up to Length to 35 amino acids, length to 40 amino acids, length to 45 amino acids, length to 50 amino acids, length to 60 amino acids, range In the length of 10-60 amino acids, in the range of 15-60 amino acids, in the range of 20-60 amino acids, in the range of 25-60 amino acids, in the range of 30 The length of -60 amino acids is in the range of 35-60 amino acids, the range is in the range of 40-50 amino acids, the range is in the range of 45-60 amino acids, the range is in the range of 10-40 The length of each amino acid ranges from 15-40 amino acids, the length ranges from 20-40 amino acids, the length ranges from 25-40 amino acids, and the range ranges from 30-40 amines The length of the base acid ranges from 35-40 amino acids, the length ranges from 10-30 amino acids, the range ranges from 15-30 amino acids, and the range ranges from 20-30 amino acids The length is in the range of 25-30 amino acids, in the range of 10-20 amino acids, or in the range of 10-15 amino acids. 8. Masking part (MM)

As described herein, the AA and BAA of the present disclosure include the pro-domain, which includes MM.

In some embodiments, the MM is selected for use with specific antibodies or antibody fragments.

In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, any natural binding partners of MM and AB contain no or substantially no similarity. In some embodiments, the similarity of any natural binding partners of MM and AB is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, or 80%. In some embodiments, the identity of any natural binding partner of MM and AB is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, or 80%. In some embodiments, the MM has no more than 50% identity with any natural binding partner of AB. In some embodiments, the MM has no more than 25% identity with any natural binding partner of AB. In some embodiments, the MM has no more than 20% identity with any natural binding partner of AB. In some embodiments, the MM has no more than 10% identity with any natural binding partner of AB.

The exemplary MM of the present disclosure may have a length of up to 15 amino acids, a length of up to 20 amino acids, a length up to 25 amino acids, a length up to 30 amino acids, up to 35 amino acids in length, up to 40 amino acids in length, up to 45 amino acids in length, up to 50 amino acids in length, up to 60 amino acids in the range of 10-60 amino acids in length, ranging from 15-60 amino acids in length, 20-60 amino acids in length, 25-60 amino acids in length and 30- The length of 60 amino acids, the range of 35-60 amino acids, the range of 40-50 amino acids, the range of 45-60 amino acids, the range of 10-40 The length of the amino acid ranges from 15-40 amino acids, the range ranges from 20-40 amino acids, the range ranges from 25-40 amino acids, and the range ranges from 30-40 amino groups The length of the acid is in the range of 35-40 amino acids, the length is in the range of 10-30 amino acids, the length is in the range of 15-30 amino acids, and the range is in the range of 20-30 amino acids Length, length in the range of 25-30 amino acids, length in the range of 10-20 amino acids, or length in the range of 10-15 amino acids, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

Exemplary MMs of this disclosure are provided in Tables 3, 7 and 8 above. 9. Linker

In many embodiments, it is desirable to insert one or more linkers, such as flexible linkers, in one or more MM-CM links, CM-AB/CM in the AA/BAA construct -scFv links, or both, provide flexibility. For example, AB, MM, and/or CM may not contain a sufficient number of residues (eg, Gly, Ser, Asp, Asn, especially Gly and Ser) to provide the desired flexibility. Thus, the ability of such BAA constructs to remain intact or activated (as disclosed herein) can benefit from the introduction of one or more amino acids to provide flexible linkers.

其中MM、CM和AB如上所界定，其中L1和L2係各獨立地並且可選地存在或不存在，且係相同或不同的撓性連接子，其包括至少1個撓性胺基酸(例如Gly、Ser)。For example, in some embodiments, AA includes one of the following formulas (wherein the following formulas represent the amino acid sequence in the N-C-terminal direction or the C-N-terminal direction):

Where MM, CM and AB are as defined above, where L1 and L2 are each independently and optionally present or absent, and are the same or different flexible linkers, which include at least one flexible amino acid (eg Gly, Ser).

In some embodiments, the BAA includes 2 heavy chains, each of which includes the structure arrangement of N-terminal to C-terminal of MM2-CM2-AB2-AB1 HC; and two light chains, each including N of MM1-CM1-AB1 LC End-to-end structure arrangement.

In some embodiments, the structure including the linker is provided in FIG. 4.

In some embodiments, (MM2)-L1-(CM2)-L2-(AB2) is linked to the heavy chain of AB1, and AB2 is scFv.

Linkers suitable for use in compositions as described herein are generally those that provide flexibility of modified AB or AA to promote inhibition of AB binding to the target. Such linkers are often referred to as flexible linkers. Suitable linkers can be easily selected and can be of any suitable different lengths, such as 1 amino acid (eg Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amines Acid to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amines Base acid to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , Or 20 amino acids. In some embodiments, suitable linkers may be 4 to 25 amino acids in length. In some embodiments, suitable linkers may be 5 to 25 amino acids in length. In some embodiments, suitable linkers may be 4 to 20 amino acids in length. In some embodiments, suitable linkers can be 5 to 20 amino acids in length.

Exemplary linkers include glycine polymer (G)n, glycine-serine polymer (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 88), and (GGGS)n ( SEQ ID NO: 89), where n is an integer of at least one), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral chains between components. Glycine has a larger phi-psi space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linker lines are provided in Table 9-1.

Those skilled in the art will understand that the design of AA may include all or part of the flexible connector, such that the connector may include flexible connectors and one or more structures that impart less flexibility to provide the desired AA structure section. 10. Conjugation

In some embodiments, the antibody, or AB of the AA, and BAA are conjugated to one dose. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable part. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a non-truncable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid disrupting agent, such as a DNA alkylating agent or DNA intercalating agent, or other DNA disrupting agent. In some embodiments, the linker is a linker that can be truncated. In some embodiments, the agent is selected from the group listed in Table 10. Table 10 : Exemplary pharmaceutical agents for conjugation

Those skilled in the art will understand that various possible moieties can be coupled to the resulting antibodies, AA, and BAA of the present disclosure. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Karger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

In some embodiments, the antibody, AA or BAA contains a detectable portion. In some embodiments, the detectable portion is a diagnostic agent.

In some embodiments, the antibody, AA or BAA contains one or more disulfide bonds. In some embodiments, the antibody, AA or BAA contains one or more lysine. In some embodiments, the antibody, AA, or BAA may be engineered to include one or more disulfide bonds or may be engineered to be position-specifically conjugated. 11. Manufacturing

The present disclosure also provides isolated nucleic acid molecules encoding antibodies, AA or BAA as described herein, and vectors including these isolated nucleic acid sequences. The present disclosure provides a method of making antibodies, AA or BAA by culturing cells under conditions that result in the expression of the antibodies, AA or BAA, wherein the cells contain this nucleic acid molecule.

In some embodiments, the cell contains this vector. In some embodiments, the vector is pLW307. In some embodiments, the vector is pLW291. In some embodiments, the vector is pEF1049. In some embodiments, the vector is pEF1050. In some embodiments, the vector is pEF1107. In some embodiments, the vector is pEF1052. (I vector system described and these sequences are provided in Example 1 below) 12. The antibody, use AA, bispecific antibodies and the BAA

In some embodiments, the antibody/bispecific antibody/AA/BAA can be used as a therapeutic agent. Such agents will generally be used to treat, alleviate and/or prevent an individual's disease or pathology. The therapeutic dosing regimen is implemented by using standard methods to identify individuals, such as individuals suffering from a disorder (or risk of developing a disease), such as human patients or other mammals.

Antibody/bispecific antibody/AA/BAA administration can eliminate or inhibit or interfere with the communication function of one or more targets.

It should be understood that the administration of the therapeutic entity according to the present disclosure will be administered together with suitable carriers, excipients, and other agents incorporated into the modulator to provide improved transfer, delivery, tolerance, etc. Many suitable modulators can be found in the pharmacopoeia known to all medicinal chemists: Remington’s Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), in particular Chapter 87 of Blaug, Seymour. These modulators include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (eg Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and Water-in-oil emulsion, carbomer wax (polyethylene glycol of various molecular weight) emulsion, semi-solid gel, and semi-solid mixture containing carbomer wax. As long as the active ingredient in the preparation is not deactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration, any of the aforementioned mixtures can be applied to the treatment and therapy according to the present disclosure. For other information about modulators, excipients and carriers that pharmaceutical chemists are familiar with, see also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 ( 2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89 (8): 967-78 (2000), Powell et al., “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52: 238-311 (1998) and in this article Quotation.

Generally, the reduction or treatment of a disease or condition involves reducing one or more symptoms or medical problems associated with the disease or condition. For example, in the case of cancer, the therapeutically effective amount of the drug can achieve one or more of the following: reduce the number of cancer cells; reduce the size of the tumor; inhibit (ie, reduce and/or stop to some extent) the cancer cells to the surrounding organs Infiltration; inhibit tumor metastasis; inhibit tumor growth to a certain extent; and/or alleviate one or more symptoms associated with cancer to some extent. In some embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in an individual, such as a human or other mammal (such as a non-human primate, companion animal (eg, cat, dog, horse), farm Animals, working animals, or zoo animals). The terms subject and patient are used interchangeably herein.

The therapeutically effective amount of the antibody/bispecific antibody/AA/BAA disclosed herein generally relates to the amount required to achieve therapeutic purposes.

As a non-limiting example, the general range of therapeutically effective doses of the antibody/bispecific antibody/AA/BAA of the present disclosure may be about 0.1 mg/kg body weight to about 50 mg/kg body weight. A common dosing frequency may range, for example, from twice a day to once a week.

Combine any known method for diagnosing or treating a specific disease to determine the effectiveness of the treatment. Methods of screening for antibodies/bispecific antibodies/AA/BAA with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known in the art.

Other covered uses include diagnostic, imaging, predictive, and detection uses. In some embodiments, antibodies/bispecific antibodies/AA/BAA are used in methods known in the art related to the positioning and/or quantification of targets (eg, for measuring one or more of a suitable physiological sample Target level, used for diagnostic methods, for protein imaging, etc.).

In some embodiments, antibodies/bispecific antibodies/AA/BAA are used to isolate one or more targets by standard techniques (eg, immunoaffinity, chromatography, or immunoprecipitation). Antibodies, AA, bispecific antibodies or BAA can be used diagnostically to monitor protein levels in tissues as part of clinical testing procedures, for example, to determine the efficacy of a given treatment regimen. The detection can be facilitated by coupling (ie, physically connecting) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase, etc.; examples of suitable prosthetic group complexes include streptavidin/biotin And avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescent isothiocyanate, rose red, dichlorotriazinylamine fluorescein ), dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin ), and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

In yet another embodiment, antibodies directed against two or more targets, bispecific antibodies, AA, BAA can be used as reagents to detect the presence of one or more targets (or fragments thereof) in the sample. In some embodiments, the antibody contains a detectable label. There are multiple strains of the resistance system, or a single strain in some embodiments. Use intact antibodies or fragments thereof (eg Fab, scFv, or F(ab') ₂ ). The term “(labeled)” with respect to a probe or antibody is intended to cover the direct labeling of the probe or antibody by coupling (ie, physically connecting) a detectable substance to the probe or antibody, and Probes or antibodies are indirectly labeled by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescent-labeled secondary antibody and end-labeling of the antibody with biotin so that it can be detected with fluorescent-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from an individual, as well as tissues, cells and fluids present in the individual. Therefore, the use of the term "biological sample" includes blood and parts or components of blood, including serum, plasma or lymph fluid. That is, the detection method of the present disclosure can be used to detect proteins in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The procedure for performing the immunoassay is described in, for example, the following: "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JR Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into individuals. For example, the antibody can be labeled with a radioactive marker, which can be detected by standard imaging techniques for its presence and location in the individual.

The antibodies, bispecific antibodies, AA, and bispecific antibodies of the present disclosure can also be used in various diagnostic and prophylactic preparations. In one embodiment, the antibody, AA, bispecific antibody, and BAA are administered to patients at risk of developing one or more of the above diseases. Genotypes, serological or biochemical markers can be used to determine the susceptibility of a patient or organ to one or more diseases.

In another embodiment of the present disclosure, antibodies, AA, bispecific antibodies, and BAA are administered to human individuals diagnosed with clinical indications related to one or more of the above diseases. Once diagnosed, antibodies, AA, bispecific antibodies, BAA are administered to reduce or reverse the clinical indications.

Antibodies, bispecific antibodies, AA, and bispecific antibodies can also be used to detect one or more targets in patient samples, and thus can be used as diagnostic reagents. For example, the antibodies, bispecific antibodies, AA, and bispecific antibodies of the invention are used in in vitro assays, such as ELISA, to detect one or more target levels in patient samples.

In one embodiment, the antibody, AA, bispecific antibody, and BAA are immobilized on a solid support (eg, a well of a microtiter plate). The immobilized antibody and/or AA is used as a capture antibody and can be used to test any target that may be present in the sample. Before contacting the immobilized antibody/AA with the patient sample, the solid support should be rinsed and treated with a blocking agent (such as milk protein or albumin) to prevent non-specific adsorption of the analyte.

Subsequently, the well is treated with a test sample suspected of containing antigen or a solution containing a standard amount of antigen. Such a sample is, for example, a serum sample from an individual who is suspected of having circulating antigen levels and is considered to be able to diagnose pathology. After rinsing out the test sample or standard solution, the solid phase support is treated with a detectably labeled secondary antibody. The labeled secondary antibody is used as a detection antibody. The level of the detectable marker is measured, and the concentration of the target antigen in the test sample is determined by comparison with the standard curve drawn from the standard sample.

It should be understood that based on the results obtained using antibodies, AA, bispecific antibodies, BAA in in vitro diagnostic assays, it is possible to stage disease in individuals based on the performance level of the target antigen. For a given disease, blood samples are collected from individuals who are diagnosed at various stages of disease progression and/or at various points in the treatment of the disease. Using a sample population that provided statistically significant results at each stage of progression or treatment, a range of antigen concentrations that could be considered as a characteristic of each stage was determined.

Antibodies, bispecific antibodies, AA and BAA can also be used in diagnostic and/or imaging methods. In some embodiments, these methods are in vitro methods. In some embodiments, these methods are in vivo methods. In some embodiments, these methods are in situ methods. In some embodiments, these methods are ex vivo methods. For example, AA and bispecific antibodies with enzymatically cleavable CM can be used to detect the presence of enzymes capable of cleaving CM. Such AA and bispecific antibodies can be used in diagnostics, which can include in vivo detection of enzyme activity (or in some embodiments, an environment with increased reduction potential that can be provided to reduce disulfide Bond, for example, can provide an environment of reduced enzyme activity (qualitative or quantitative)), which is obtained by measuring activation or bispecific activation of antibodies in a given cell or tissue of a given host organism (ie, cleavage from AA or BAA) Of antibodies or bispecific antibodies). This accumulation of activated bispecific antibodies not only indicates that the tissue exhibits enzyme activity (or an increased reduction potential depending on the nature of the CM), but also indicates that the tissue exhibits at least one target to which the activated bispecific antibody binds.

For example, CM can be selected as a protease substrate for proteases found at tumor sites, at viral or bacterial infection sites, at biologically restricted sites (eg, in abscesses, organs), and the like. At least one of the ABs can bind to the target antigen. Using methods familiar to those skilled in the art, detectable labels (eg, fluorescent labels or radioactive labels or radiotracers) can be conjugated to antibodies, AA, bispecific antibodies, BAA's AB, or other regions. Suitable detectable labels are discussed in the context of the above screening methods, and additional specific examples are provided below. Use at least one AB that is specific for a disease state protein or peptide and a protease with enhanced activity in the target disease tissue, relative to where the CM-specific enzyme is not present at a detectable level or is lower than the level in disease tissue Or tissues that are inactive (eg, in the form of zymogens or complexed with inhibitors), AA will show an increased rate of binding to diseased tissues. Because the kidney filtration system quickly removes small proteins and peptides from the blood, and because CM-specific enzymes are not present at detectable levels (or at lower levels in non-disease tissues or in an inactive conformation), Compared to non-disease tissues, the accumulation of activated bispecific antibodies in diseased tissues is enhanced.

In another example, the disclosed antibodies, antibodies/bispecific antibodies/AA/BAA can be used to detect the presence or absence of a cleaving agent in a sample. For example, in the case where the antibody/bispecific antibody/AA/BAA contains CM that is easily cleaved by the enzyme, the BAA can be used to detect (qualitatively or quantitatively) the presence of the enzyme in the sample. In another example, if the antibody/bispecific antibody/AA/BAA contains CM that is easily cleaved by the reducing agent, the antibody/bispecific antibody/AA/BAA can be used (qualitatively or quantitatively) to detect the reducing conditions in the sample exist. To facilitate analysis in these methods, the antibody/bispecific antibody/AA/BAA can be detectably labeled and can be bound to a support (eg, a solid support, such as a slide or bead). The detectable label may be on a portion of the antibody/bispecific antibody/AA/BAA that is not released after cleavage. For example, the detectable label may be a quenched fluorescent label or other label that is not detectable before cleavage occurs. For example, the immobilized, detectably labeled antibody/bispecific antibody/AA/BAA can be contacted with a sample suspected of containing enzymes and/or reducing agents for sufficient time for lysis to occur, and then washed to remove excess samples and contamination Thing. The presence or absence of a cleaving agent (such as an enzyme or reducing agent) in the sample is then assessed by the change in the detectable signal of antibody/bispecific antibody/AA/BAA before contact with the sample, for example, cleavage in the sample The presence and/or increase of a detectable signal caused by the cleavage of the antibody/bispecific antibody/AA/BAA by the agent.

Such a detection method may be adapted to provide detection of the presence or absence of a target capable of binding at least one AB of the antibody/bispecific antibody/AA/BAA of the present disclosure. Therefore, the assay can be adapted to assess the presence or absence of a cutting agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence and/or increase of the detectable label of the antibody/bispecific antibody/AA/BAA as described above, and the presence or absence of the target can be detected by The target AB complex is detected, for example, by using a detectably labeled anti-target antibody.

The disclosed AA/BAA can also be used for in situ imaging to verify AA activation, for example, by protease cleavage and binding to specific targets. In situ imaging is a technique capable of localizing proteolytic activity and targets in biological samples, such as cell cultures or tissue sections. Using this technique, binding to a given target and proteolytic activity can be confirmed based on the presence of a detectable label (eg, fluorescent label).

These techniques are useful for any frozen cells or tissues (such as tumor tissues) or healthy tissues derived from the disease site. These techniques are also useful for fresh cell or tissue samples.

In these technologies, AA/BAA is marked by a detectable mark. The detectable label can be a fluorescent dye (eg, fluorophore, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), Alexa Fluor® label), near infrared (NIR) Dyes (e.g. Qdot® nanocrystals), colloidal metals, haptens, radiolabels, biotin and amplification reagents (e.g. streptavidin) or enzymes (e.g. horseradish peroxidase or alkaline phosphatase) .

Testing the label in a sample that has been incubated with labeled AA or BAA indicates that the sample contains a target and contains a protease specific for the CM of the disclosed AA or BAA. In some embodiments, a broad-spectrum protease inhibitor (eg, an inhibitor described herein) can be used and/or by using an agent specific for a protease (eg, specific for protease interstitial protease (MT-SP1) Antibodies (eg A11)) to confirm the presence of proteases and inhibit the proteolytic activity of MT-SP1; see for example International Patent No. WO 2010/129609, published on November 11, 2010. The same method using broad-spectrum protease inhibitors (such as those described herein) and/or by using more selective inhibitors can be used to identify proteases or protease classes specific for the CM of the AA or BAA of the present disclosure. In some embodiments, a target-specific reagent can be used to confirm the presence of the target, or a detectable label can compete with an unlabeled target. In some embodiments, unlabeled AA can be used for detection by a labeled secondary antibody or a more complex detection system.

Similar techniques can also be used for in vivo imaging, where fluorescent signals detected in individuals (eg, mammals including humans) indicate that the disease site contains a target and contains CM for the AA or BAA of the present disclosure Specific protease.

These techniques can also be used in kits and/or as reagents for identifying or characterizing protease activity in a variety of cells, tissues, and organisms based on protease-specific CM detection in AA or BAA of the present disclosure. 13. Therapeutic administration

It should be understood that the administration of the therapeutic entity according to the present disclosure will be administered together with suitable carriers, excipients, and other agents incorporated into the modulator to provide improved transfer, delivery, tolerance, etc. Many suitable modulators can be found in the pharmacopoeia known to all medicinal chemists: Remington’s Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), in particular Chapter 87 of Blaug, Seymour. These modulators include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (eg Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and Water-in-oil emulsion, carbomer wax (polyethylene glycol of various molecular weight) emulsion, semi-solid gel, and semi-solid mixture containing carbomer wax. As long as the active ingredient in the preparation is not deactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration, any of the aforementioned mixtures can be applied to the treatment and therapy according to the present disclosure. For other information about modulators, excipients and carriers that pharmaceutical chemists are familiar with, see also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 ( 2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89 (8): 967-78 (2000), Powell et al., “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52: 238-311 (1998) and in this article Quotation.

In some embodiments, the antibody, bispecific antibody, AA, or BAA (or its conjugated composition) is administered in combination with one or more other agents or combinations of other agents. Suitable other agents include current drugs and/or surgical therapies for the intended application. For example, they can be used in combination with additional chemotherapeutic or antitumor agents.

In some embodiments, the disclosed antibody, bispecific antibody, AA, or BAA (or its conjugated composition) is administered in combination with one or more other agents selected from the group consisting of: Antibody, conjugated antibody, AA, conjugated AA, bispecific antibody, conjugated bispecific antibody, BAA, or conjugated BAA. In some embodiments, the antibody portion of any of the other agents referenced above is directed to a target, such as one or more targets disclosed in Table 9. It should be understood that in some embodiments, the antibody portion of the disclosed antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) and the antibody portion of other agents are directed to the same target (e.g., Both can target EGFR). In some embodiments, they target the same target but different epitopes. In some embodiments, they are completely targeted to different targets (for example, the activatable antibody of the present invention targeting EGFR can be administered in combination with AA targeting different targets; likewise, for example, targeting EGFR and CD3 The disclosed BAA can be co-administered with AA targeting different targets.

In some embodiments, the disclosed antibody, bispecific antibody, AA or BAA (or conjugated composition thereof) is administered in combination with an immunotherapeutic agent. In some embodiments, the disclosed antibody, bispecific antibody, AA or BAA (or conjugated composition thereof) is administered in combination with a chemotherapeutic agent. In some embodiments, the disclosed antibody, bispecific antibody, AA or BAA (or conjugated composition thereof) is administered in combination with both immunotherapeutic agents and chemotherapeutic agents. In some embodiments, any of these combination embodiments is administered with one or more other agents.

In some embodiments, they are formulated as a single therapeutic composition, and the antibody/bispecific antibody/AA/BAA and other agents are administered simultaneously. Alternatively, the antibody/bispecific antibody/AA/BAA is administered separately from each other, for example, they are formulated as a separate therapeutic composition, and the antibody/bispecific antibody/AA/BAA and other agents are administered simultaneously, or In the treatment regimen, the antibody/bispecific antibody/AA/BAA and other agents are administered at different times. Antibodies/bispecific antibodies/AA/BAA and other agents can be administered multiple times.

The antibody/bispecific antibody/AA/BAA can be incorporated into a pharmaceutical composition suitable for administration. The principles and precautions for preparing such compositions, as well as guidelines for component selection are provided in, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co ., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such a composition usually includes an antibody/bispecific antibody/AA/BAA and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration . Suitable carriers are described in the latest version of Remington’s Pharmaceutical Sciences, which is a standard reference in the art, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, it is considered to be used in the composition.

Preparations for in vivo administration must be sterile. Filtration through sterile filter membranes is easy to achieve.

The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of administration routes include parenteral, such as intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include the following ingredients: sterile diluents, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; Bacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphoric acid Salt, as well as agents for adjusting tonicity, such as sodium chloride or glucose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (when water-soluble) or dispersions, and sterile powders for the temporary preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should have a certain degree of fluidity so that it is easy to inject. It must be stable under production and storage conditions, and must be preservative treated to prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyhydric alcohols (such as glycerin, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the required particle size and by using surfactants. Various antibacterial agents and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, sodium thiomersal, etc., can be used to prevent the action of microorganisms. In many cases, it is suitable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. By including agents that delay absorption in the composition, such as aluminum monostearate and gelatin, prolonged absorption of the injectable composition can be achieved.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired ingredients from their previously sterile filtered solution.

Oral compositions usually contain an inert diluent or an edible carrier. They can be filled into gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be combined with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is administered orally and wiped dry and spit out or swallowed. Pharmaceutically compatible adhesives and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, such as microcrystalline cellulose, gums, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrate Agents, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as mint, salicylic acid Methyl acid or orange flavoring.

For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressure vessel or dispenser that contains a suitable propellant, such as a gas such as carbon dioxide or a nebulizer.

Systemic administration can also be performed via mucosal or percutaneous methods. For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are well known in the art, and include, for example, detergents for transmucosal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, ointment, gel, or emulsion, as is generally known in the art.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enema forms for rectal delivery.

In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as sustained/controlled release formulations, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The preparation method of such formulations will be obvious to those skilled in the art.

For example, the active ingredient may be embedded in microcapsules prepared by, for example, coagulation technology or by interfacial polymerization, the microcapsules are respectively in a colloidal drug delivery system (eg, respectively in a colloidal drug delivery system (eg, liposome, albumin Microspheres, microemulsions, nanoparticles and nanocapsules) or hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in large emulsions).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, eg films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (US Patent 3,773,919), L-glutamic acid, and gamma -Ethyl-L-glutamine copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ^TM (from lactic acid-glycolic acid copolymer and leuprolide acetate Lin composed of injectable microspheres) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels can release proteins in a shorter time.

This material is also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 4,522,811.

It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose of a subject to be treated; each unit contains a predetermined amount of active compound, which can be calculated to produce the desired together with the desired pharmaceutical carrier treatment effect. The specification of the dosage unit form of the present disclosure is directly determined by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technical field of formulating this active compound for the treatment of this individual disease.

The pharmaceutical composition can be included in a container, package or dispenser together with instructions for administration.

For the specific indication being treated, the formulation may also contain more than one active compound, for example, those with complementary activities that do not adversely affect each other. Or in addition, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably combined in an amount effective for the intended purpose.

In one embodiment, the active compound is administered in combination therapy, ie, in combination with other agents, such as therapeutic agents, for the treatment of pathological conditions or disorders, such as autoimmune diseases and inflammatory diseases. As used herein, the term "in combination" refers to the administration of agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds can still be detected at an effective concentration at the treatment site when the second compound is administered.

For example, the combination therapy may include one or more antibodies/bispecific antibodies/AA/BAA of the invention co-formulated and/or co-administered with one or more additional therapeutic agents, additional therapeutic agents such as one or more cytokines And growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below. In addition, one or more antibodies/bispecific antibodies/AA/BAA described herein can be used in combination with two or more therapeutic agents described herein (eg, one BAA is administered with another BAA or AA disclosed herein) ,and many more). Such combination therapy can advantageously utilize lower doses of the administered therapeutic agent, thereby avoiding possible toxicity or complications associated with various monotherapy.

In other embodiments, one or more antibodies of the present disclosure may be co-formulated and/or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. Non-limiting examples of drugs or inhibitors that can be used in combination with the antibodies described herein include, but are not limited to, one or more: non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, tenidap, naproxen, or Loxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids, such as prednisolone; cytokine inhibitory anti-inflammatory drugs (CSAID); nucleotide biosynthesis inhibition Agents, such as purine biosynthesis inhibitors, folic acid antagonists (such as methotrexate (methotrexate) (N-[4-[[(2,4-diamino-6-pyridyl)methyl]methylamino ]Benzyl]-L-glutamic acid); and inhibitors of pyrimidine biosynthesis, such as dihydroorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the present disclosure These include NSAID, CSAID, (DHODH) inhibitors (eg leflunomide) and folate antagonists (eg methotrexate).

Examples of additional inhibitors include one or more of the following: corticosteroids (oral, inhalation, and local injection); immunosuppressive agents, such as cyclosporine, tacrolimus (FK-506); and mTOR inhibitors, such as Rolimus (rapamycin-RAPAMUNE ^™ or rapamycin derivatives, for example soluble rapamycin derivatives (for example, ester rapamycin derivatives, for example CCI-779); by pro-inflammatory cytokines For example, drugs that interfere with signaling by TNFα or IL-1 (such as IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors, such as celecoxib, rofecoxib, and their variants Phosphodiesterase inhibitors, such as R973401 (Phosphodiesterase type IV inhibitors); Phospholipase inhibitors, such as cytosolic phospholipase 2 inhibitors (cPLA2) (such as trifluoromethyl ketone analogs); blood vessels Inhibitors of endothelial cell growth factor or growth factor receptors, such as VEGF inhibitors and/or VEGF-R inhibitors; and angiogenesis inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the present disclosure are immunosuppressants, such as Cyclosporine, tacrolimus (FK-506); mTOR inhibitors, such as sirolimus (rapamycin) or rapamycin derivatives, such as soluble rapamycin derivatives (such as ester rapamycin) Derivatives such as CCI-779); COX2 inhibitors such as celecoxib (celecoxib) and its variants; phospholipase inhibitors such as cytoplasmic phospholipase 2 (cPLA2) inhibitors such as trifluoromethyl Ketone analogs.

Other examples of therapeutic agents that can be combined with the antibodies of the present disclosure include one or more of the following: 6-mercaptopurine (6-MP); azathioprine (azathioprine) sulfasalazine; mesalamine (mesalamine); Olsalazine; chloroquine/hydroxychloroquine (PLAQUENIL ^® ); pencillamine; aurothiornalate (intramuscular and oral); azathioprine; azathioprine; coichicine ; Β-2 adrenergic receptor agonists (salbutamol (salbutamol), terbutaline (terbutaline), salmeterol (salmeteral)); xanthine (theophylline, anorophylline); cromoglycate (cromoglycate); Nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonist; antithrombotic agent; complement Inhibitors; and adrenergic drugs.

In some embodiments, the disclosed antibodies/bispecific antibodies/AA/BAA can be combined with one or more antibodies/bispecific antibodies/AA/BAA. 14. Set

Provided herein is a set comprising any one or more of the antibodies, AA, bispecific antibodies, and BAA provided herein.

The kit may include any one or more of the antibodies, AA, bispecific antibodies, and BAA provided herein in a composition suitable for storage or transportation. The kit may contain a container, diluent, solvent, second composition, or any component suitable for converting the storage or transportation composition into a composition suitable for the method disclosed herein; the method may be For example, the treatment methods disclosed herein. The kit can contain instructions for use. Illustrative implementation

The invention can be defined with reference to the following illustrative implementations:

Embodiment 1. A bispecific activatable antibody (BAA), wherein the BAA specifically binds to two targets when activated, and wherein the BAA contains the following structure when not activated: a) Specificity IgG antibody (AB1) bound to the first target, wherein the AB1 contains: i. two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. two first pre-domains, each of which contains The first masking portion (MM1) connected to the first cleavable portion (CM1) in the direction from N-terminus to C-terminus, wherein the carboxy terminus of each first pre-domain is connected to the amine terminus of each light chain of the AB1 Ligation, where-the MM1 reduces the binding of the AB1 to its target; and-the CM1 polypeptide, which is used as a substrate for the first protease, b) two scFv (AB2), each of which specifically binds CD3 _Ɛ , Each AB2 contains: i. A light chain variation region (VL) connected to a heavy chain variation region (VH), wherein the carboxyl end of each AB2 is connected to the amine end of each of the AB1 heavy chains, where- VL contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH contains the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and-the VL is borrowed Linked to the VH by linker L3, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and ii. two second pre-domains, which Each includes a second masking portion (MM2) connected to the second cleavable portion (CM2) in the direction from N-terminal to C-terminal, wherein the carboxyl terminal of each second pre-domain is connected to the amine terminal of each AB2, wherein - the MM2 and reduce binding of the _CD3 ɛ of AB2; - MM2 comprising the amino acid sequence of the group consisting of the following selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and-the CM2 polypeptide is used as a substrate for the second protease.

Embodiment 2. The BAA as in embodiment 1, wherein the VL is connected to the VH by a linker including the amino acid sequence SEQ ID NO: 98.

Embodiment 3. The BAA as in embodiment 1, wherein the VL is connected to the VH by a linker comprising the amino acid sequence SEQ ID NO: 108.

Embodiment 4. The BAA according to any one of Embodiments 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 12.

Embodiment 5. The BAA according to any one of Embodiments 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 105.

Embodiment 6. The BAA according to any one of Embodiments 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 106.

Embodiment 7. The BAA according to any one of Embodiments 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 107.

Embodiment 8. The BAA according to any one of Embodiments 1 to 7, wherein the AB1 binds to a tumor target.

Embodiment 9. The BAA according to any one of Embodiments 1 to 8, wherein the AB1 binds to EGFR.

Embodiment 10. The BAA according to any one of Embodiments 1 to 9, wherein the MM1 comprises an amino acid sequence selected from the group consisting of the sequences presented in Table 7 or Table 8.

Embodiment 11. The BAA according to any one of Embodiments 1 to 10, wherein the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 85.

Embodiment 12. The BAA according to any one of Embodiments 1 to 11, wherein the MM1 comprises the amino acid sequence SEQ ID NO: 78.

Embodiment 13. The BAA according to any one of Embodiments 1 to 11, wherein the MM1 comprises the amino acid sequence SEQ ID NO: 85.

Embodiment 14. The BAA according to any one of Embodiments 1 to 13, wherein the CM1 or CM2 comprises the amino acid sequence of any one of the CMs listed in Table 4 or Table 4-1.

Embodiment 15. The BAA according to any one of Embodiments 1 to 14, wherein the CM1 or CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.

Embodiment 16. The BAA according to any one of Embodiments 1 to 15, wherein the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.

Embodiment 17. The BAA according to any one of Embodiments 1 to 15, wherein the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17.

Embodiment 18. The BAA according to any one of Embodiments 1 to 17, wherein the AB1 includes an Fc region including amino acid positions L234, L235, N297, and P331 (indexed by EU shown in Kabat The amino acid substitution of at least one of the numbers) makes the BAA have a reduced effector function.

Embodiment 19. The BAA of Embodiment 18, wherein the AB1 includes an amino acid substitution in at least two of amino acid positions L234, L235, and P331.

Embodiment 20. The BAA of any one of Embodiments 18 to 19, wherein the AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P331.

Embodiment 21. The BAA of any one of Embodiments 18 to 20, wherein the AB1 includes amino acid substitutions of L234F, L235E, and P331S.

Embodiment 22. The BAA according to any one of Embodiments 1 to 21, wherein the AB1 includes an Fc region, which includes an amino acid substitution in N297.

Embodiment 23. The BAA of any one of Embodiments 18 to 22, wherein the AB1 comprises amino acid substitutions of L234F, L235E, P331S, and N297Q.

Embodiment 24. The BAA according to any one of Embodiments 18 to 23, wherein the heavy chain of AB1 comprises the amino acid sequence shown in Table 4-2 selected from the group consisting of: SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122.

Embodiment 25. The BAA according to any one of Embodiments 18 to 23, wherein the heavy chain of AB1 comprises the amino acid sequence shown in Table 6 selected from the group consisting of: SEQ ID NO: 69. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76.

Embodiment 26. The BAA according to any one of Embodiments 1 to 7 and 10 to 25, wherein the first target is selected from the group consisting of the targets presented in Table 9.

Embodiment 27. The BAA according to any one of Embodiments 1 to 26, wherein the first and second front structures are based on the formula (MM)-L1-(CM)-L2 in the direction from the N terminal to the C terminal Each domain includes a linker L1 and a linker L2.

Embodiment 28. A BAA (CI138), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 155 or SEQ ID NO: 124, and shown in SEQ ID NO: 132 or SEQ ID NO: 140 The light chain amino acid sequence.

Embodiment 29. A BAA (CI139), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 157 or SEQ ID NO: 126, and shown in SEQ ID NO: 132 or SEQ ID NO: 140 The light chain amino acid sequence.

Embodiment 30. A BAA (CI158), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 161 or SEQ ID NO: 128, and shown in SEQ ID NO: 132 or SEQ ID NO: 140 The light chain amino acid sequence.

Embodiment 31. A BAA (CI140), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 159 or SEQ ID NO: 25, and shown in SEQ ID NO: 132 or SEQ ID NO: 140 The light chain amino acid sequence.

Embodiment 32. An activatable antibody (AA), wherein the AA specifically binds to a target when activated, and wherein the AA contains the following structure when not activated: a) at least one scFv, which comprises a light chain variant region (VL) connected to a heavy chain variant region (VH), wherein the VL is connected to the VH by a linker L3, the linker comprises a member selected from the group consisting of SEQ ID NO: The amino acid sequence of the group consisting of 98 and SEQ ID NO: 108; and b) Prodomain, which contains: i. a masking moiety (MM) coupled to the scFv, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the scFV to its target; and ii. A truncable portion (CM) coupled to the scFv, wherein the CM is a substrate polypeptide used as a protease.

Implementation aspect 33. AA as in implementation aspect 32, wherein the target of the scFv is CD3 _Ɛ .

34. An embodiment of aspects of activating antibody (AA), wherein upon activation of the AA by the specific binding to the target, and wherein the AA comprises the following structure when non-activated: a) specifically binds to the _CD3 Ɛ Antibody or antigen-binding fragment (AB); and b) Prodomain (prodomain), which includes: i. Masking portion (MM) coupled to the AB, where the AA is in an uncut state , The MM reduces or inhibits the binding of the AB and CD3 _Ɛ , wherein the MM contains the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and ii. The coupling with the AB may The cut part (CM), wherein the CM is used as a substrate for proteases.

Embodiment 35. The AA of any one of Embodiments 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13-17.

Embodiment 36. The AA according to any one of Embodiments 32 to 34, wherein the CM includes a substrate that can be cut by serine protease or MMP.

Embodiment 37. 　 AA as in any one of Embodiments 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-56.

Embodiment 38. The AA according to any one of Embodiments 32 to 34, wherein the protease is MMP.

Embodiment 39. The AA according to any one of Embodiments 32 to 34, wherein the protease is serine protease.

Embodiment 40. 　 AA according to any one of claims 32 to 39, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH comprises SEQ ID NO: 2 Or the amino acid sequence shown in SEQ ID NO: 3.

Embodiment 41. The AA according to any one of Embodiments 32 to 40, wherein, according to the formula (MM)-L1-(CM)-L2 in the N-terminal to C-terminal direction, the pro-domain includes a linker L1 And linker L2.

Embodiment 42. 　 The AA or BAA of any one of Embodiments 1 to 41, wherein the antigen-binding fragment thereof is selected from the group consisting of: Fab fragment, F(ab') 2 fragment, scFv, scAb, dAb, single domain heavy chain antibodies, and single domain light chain antibodies.

Embodiment 43. 　 The AA or BAA of any one of Embodiments 1 to 42, wherein the anti-systemic rodent antibody, chimeric antibody, humanized antibody, or fully human monoclonal antibody.

Embodiment 44. The AA according to any one of Embodiments 1 to 43, wherein the AA is part of BAA.

Embodiment 45. A pharmaceutical composition comprising the AA or BAA of any one of Embodiments 1 to 44 and optionally a carrier.

Embodiment 46. The pharmaceutical composition according to embodiment 45, which contains other agents.

Embodiment 47. The pharmaceutical composition according to embodiment 46, wherein the other agent is a therapeutic agent.

Embodiment 48. An isolated nucleic acid molecule that encodes AA or BAA as in any one of Embodiments 1 to 44.

Embodiment 49. A vector comprising the isolated nucleic acid molecule as in Embodiment 48.

Embodiment 50. A vector comprising the nucleic acid sequence of pEF1049.

Embodiment 51. A vector comprising the nucleic acid sequence of pEF1050.

Embodiment 52. A vector comprising the nucleic acid sequence of pEF1107.

Embodiment 53. A vector comprising the nucleic acid sequence of pEF1052.

Embodiment 54. A cell comprising the vector according to any one of Embodiments 50 to 53.

Embodiment 55. A cell comprising pEF1049 and pLW246.

Embodiment 56. A cell comprising pEF1050 and pLW246.

Embodiment 57. A cell comprising pEF1107 and pLW246.

Embodiment 58. A cell comprising pEF1052 and pLW246.

Embodiment 59. 　A method of manufacturing AA or BAA according to any one of Embodiments 1 to 44 by culturing a cell under conditions that result in expression of AA or BAA, wherein the cell contains the nucleic acid according to Embodiment 48 The molecule or the vector as in any one of embodiments 49 to 53, or the cell as in any one of embodiments 54 to 58.

Embodiment 60. A method of treating, reducing symptoms, or delaying progression of a disorder or disease, which comprises treating a therapeutically effective amount of AA or BAA as in any one of Embodiments 1 to 44, or as in Embodiment 45 The pharmaceutical composition of any one of to 47 is administered to an individual in need of it.

Embodiment 61. The method of embodiment 60, wherein the disorder or disease comprises disease cells that express EGFR.

Embodiment 62. The method of any one of embodiments 60 or 61, wherein the disorder or disease is cancer.

Embodiment 63. The method according to embodiment 62, wherein the cancer is bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck Cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, squamous cell carcinoma, or skin cancer.

Embodiment 64. 　A method of inhibiting angiogenesis in an individual, comprising a therapeutically effective amount of AA or BAA as in any one of Embodiments 1 to 44, or a medicine as in any one of Embodiments 45 to 47 The composition is administered to the individual who needs it.

Embodiment 65. The method according to any one of embodiments 60 to 64, wherein the method comprises administration of other agents.

Embodiment 66. The method according to embodiment 65, wherein the other agent is a therapeutic agent.

Embodiment 67. A method of reducing damage to healthy tissue caused by antibody binding to its target on healthy tissue and on diseased tissue, the method comprising combining AA, BAA or a pharmaceutical composition containing AA or BAA When administered to an individual in need thereof, the AA or BAA is the AA or BAA of any one of Embodiments 1 to 44.

Embodiment 68. 　A method for improving the tolerance of antibody treatment, comprising administering AA, BAA, or a pharmaceutical composition containing AA or BAA to an individual in need thereof, wherein the AA or BAA is as in Embodiment 1 to Any of 44 AA or BAA.

Embodiment 69. A method of recruiting T cells to tumor tissues, which comprises administering AA or BAA or a pharmaceutical composition containing AA or BAA to an individual in need thereof, wherein the AA or BAA is as in Embodiment 1 AA or BAA to any of 44.

Embodiment 70. The AA or BAA according to any one of Embodiments 1 to 44, or the pharmaceutical composition according to any one of Embodiments 45 to 47, which is used for treatment of a disease or disease, reduction of symptoms, Or postpone progress.

Embodiment 71. The use as in embodiment 70, wherein the disorder or disease comprises disease cells expressing EGFR.

Embodiment 72. The use according to embodiment 70 or 71, wherein the disorder or disease is cancer.

实施例73. The use as in embodiment 72, wherein the cancer is bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck Cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, squamous cell carcinoma, or skin cancer.

Embodiment 74. The AA or BAA according to any one of Embodiments 1 to 44, or the pharmaceutical composition according to any one of Embodiments 45 to 47, which is a method for inhibiting angiogenesis in an individual.

Embodiment 75. The use according to any one of Embodiments 70 to 74, wherein the method includes the use of other agents.

Embodiment 76. The method according to embodiment 75, wherein the other agent is a therapeutic agent.

Embodiment 77. The AA or BAA according to any one of Embodiments 1 to 44, or the pharmaceutical composition according to any one of Embodiments 45 to 47, which is used to reduce the binding of antibodies to its healthy tissue It is also a method of damaging healthy tissues caused by targets on diseased tissues.

Embodiment 78. The AA or BAA according to any one of Embodiments 1 to 44, or the pharmaceutical composition according to any one of Embodiments 45 to 47, which is a method for improving the tolerance of antibody treatment.

Embodiment 79. The AA or BAA according to any one of Embodiments 1 to 44, or the pharmaceutical composition according to any one of Embodiments 45 to 47, which is a method for recruiting T cells to tumor tissue . Examples

The following examples are included for illustrative purposes and are not intended to limit the scope of the invention. Example 1 Antibodies, BAA and activated BAA sequences, vector construction and performance of antibodies of interest

The molecules provided in Table 11 below have been constructed and tested. As indicated, the activated molecule is manufactured in a masked form and proteolytically cut to produce an activated form.

胺基酸序列 [間隔子SEQ ID NO:133][pLW289無間隔子SEQ ID NO: 136]

核苷酸序列 [間隔子SEQ ID NO:137][pLW246無間隔子SEQ ID NO: 139]

胺基酸序列 [間隔子SEQ ID NO:138][pLW246無間隔子SEQ ID NO: 140]

CI138: CF41-2008-C225v5Fcmt4-h20GG-0011-v16(L3)sc-H-N (其中L3係SEQ ID NO: 108)

核苷酸序列 [間隔子SEQ ID NO:141][pEF1049無間隔子SEQ ID NO: 123]

胺基酸序列 [間隔子SEQ ID NO:133][pEF1049無間隔子SEQ ID NO: 124]

序列提供如上 CI139: CF41-2008-C225v5Fcmt4-hCD1-0011-v16sc-H-N pEF1050: HC hCD1-0011-v16sc-C225v5Fcmt4 (H-N) 核苷酸序列 [間隔子SEQ ID NO:141][pEF1050無間隔子SEQ ID NO: 125]

胺基酸序列 [間隔子SEQ ID NO:133][pEF1050無間隔子SEQ ID NO:126]

CI158: CF41-2008-C225v5Fcmt4-hCD1 變異體 -0011-v16sc-H-N pEF1107: HC hCD1 變異體 -0011-v16sc-C225v5Fcmt4 (H-N) 核苷酸序列 [間隔子SEQ ID NO:141][pEF1107無間隔子SEQ ID NO:127]

胺基酸序列 [間隔子SEQ ID NO:133][pEF1107無間隔子SEQ ID NO:128]

pLW246: LC CF41-2008-C225v5 序列提供如上 CI140:CF41-2008-C225v5Fcmt4-hCD13-0011-v16sc-H-N pEF1052: HC hCD13-0011-v16sc-C225v5Fcmt4 (H-N) 核苷酸序列 [間隔子SEQ ID NO:141][pEF1052無間隔子SEQ ID NO:142]

胺基酸序列 [間隔子SEQ ID NO:133][pEF1052無間隔子SEQ ID NO:25]

pLW246: LC CF41-2008-C225v5 序列提供如上(SEQ ID NO: 131及SEQ ID NO: 132) 抗 CD3 scFv 變異體 v12 輕鏈Lv12 (SEQ ID NO: 1) 重鏈HV12 (SEQ ID NO: 2) 其中連接子L3連接輕鏈及重鏈，其中L3係選自SEQ ID NO: 98及SEQ ID NO: 108。 抗 CD3 scFv 變異體 v16 輕鏈LV12 (SEQ ID NO: 1) 重鏈HV20 (SEQ ID NO: 3) 其中連接子L3連接輕鏈及重鏈，其中L3係選自SEQ ID NO: 98及SEQ ID NO: 108。 抗 CD3 scFv 變異體 v19 輕鏈LV19 (SEQ ID NO: 4) 重鏈HV20 (SEQ ID NO: 3) 其中連接子L3連接輕鏈及重鏈，其中L3係選自SEQ ID NO: 98及SEQ ID NO: 108。 抗 CD3 scFv 變異體 v26 輕鏈LV19 (SEQ ID NO: 4) 重鏈HV12 (SEQ ID NO: 2) 其中連接子L3連接輕鏈及重鏈，其中L3係選自SEQ ID NO: 98及SEQ ID NO: 108。 載體構築The sequences of molecules and vectors are provided below. Brackets indicate certain components of the molecule that are present. In some sequences, the linker is provided. The underlined amino acids represent the predicted CDR sequences.CI106: CF41-2008-C225v5Fcmt4-h20GG-0011-v16sc-HN pLW289: HC h20GG-0011-v16sc-C225v5Fcmt4 (HN) Nucleotide sequence [Spacer SEQ ID NO: 134] [pLW289 No Spacer SEQ ID NO: 135]

Amino acid sequence [Spacer SEQ ID NO: 133] [pLW289 No Spacer SEQ ID NO: 136]

Nucleotide sequence [Spacer SEQ ID NO: 137] [pLW246 without spacer SEQ ID NO: 139]

Amino acid sequence [Spacer SEQ ID NO: 138] [pLW246 without spacer SEQ ID NO: 140]

CI138: CF41-2008-C225v5Fcmt4-h20GG-0011-v16(L3)sc-HN (Wherein L3 is SEQ ID NO: 108)

Nucleotide sequence [Spacer SEQ ID NO: 141] [pEF1049 No Spacer SEQ ID NO: 123]

Amino acid sequence [Spacer SEQ ID NO: 133] [pEF1049 No Spacer SEQ ID NO: 124]

The sequence is provided above CI139: CF41-2008-C225v5Fcmt4-hCD1-0011-v16sc-HN pEF1050: HC hCD1-0011-v16sc-C225v5Fcmt4 (HN) Nucleotide sequence [Spacer SEQ ID NO: 141] [pEF1050 without spacer SEQ ID NO: 125]

Amino acid sequence [Spacer SEQ ID NO: 133] [pEF1050 No Spacer SEQ ID NO: 126]

CI158: CF41-2008-C225v5Fcmt4-hCD1 Variant -0011-v16sc-HN pEF1107: HC hCD1 Variant -0011-v16sc-C225v5Fcmt4 (HN) Nucleotide sequence [Spacer SEQ ID NO: 141] [pEF1107 No Spacer SEQ ID NO: 127]

Amino acid sequence [Spacer SEQ ID NO: 133] [pEF1107 No Spacer SEQ ID NO: 128]

pLW246: LC CF41-2008-C225v5 The sequence is provided above CI140: CF41-2008-C225v5Fcmt4-hCD13-0011-v16sc-HN pEF1052: HC hCD13-0011-v16sc-C225v5Fcmt4 (HN) Nucleotide sequence [Spacer SEQ ID NO: 141] [pEF1052 No Spacer SEQ ID NO: 142]

Amino acid sequence [Spacer SEQ ID NO: 133] [pEF1052 No Spacer SEQ ID NO: 25]

pLW246: LC CF41-2008-C225v5 The sequence is provided above (SEQ ID NO: 131 and SEQ ID NO: 132) anti- CD3 scFv Variant v12 Light chain Lv12 (SEQ ID NO: 1) Heavy chain HV12 (SEQ ID NO: 2) The linker L3 connects the light chain and the heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108. anti- CD3 scFv Variant v16 Light chain LV12 (SEQ ID NO: 1) Heavy chain HV20 (SEQ ID NO: 3) The linker L3 connects the light chain and the heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108. anti- CD3 scFv Variant v19 Light chain LV19 (SEQ ID NO: 4) Heavy chain HV20 (SEQ ID NO: 3) The linker L3 connects the light chain and the heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108. anti- CD3 scFv Variant v26 Light chain LV19 (SEQ ID NO: 4) Heavy chain HV12 (SEQ ID NO: 2) The linker L3 connects the light chain and the heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108. Carrier construction

Standard molecular biology techniques are used to clone heavy and light chains into mammalian expression vectors, respectively. Briefly, the DNA fragment encoding the region of interest is amplified with primers bound to the ends. The overlapping fragments are merged and amplified with flanking primers as needed to construct the entire desired region. The DNA fragments were then cloned into expression vectors using commercially available homologous recombination kits (MCLabs, South San Francisco, CA). The mammalian expression vector is a modified version of cDNA ^TM 3.1 (+) from Invitrogen, with a selectable marker of G418 or hygromycin. The QuikChange Kit (Agilent, Santa Clara, CA) was used to introduce mutations. Performance of AA and double-masked BAA (BAA)

AA and BAA were expressed in mammalian cells using a standard transfection kit (Life Technologies, Grand Island, NY). Briefly, 293 cells were transfected with nucleic acids using a lipid-based system according to the experimental procedures recommended by the manufacturer. AA and double-masked BAA were purified from the cell-free supernatant using protein A beads (GE, Piscataway, NJ), and concentrated using standard buffer exchange columns (Millipore, Temecula, CA). Example 2 Performance, purification, and dynamic aggregation tests of dual-masked bispecific activatable antibodies

Temporarily transfected bispecific activatable antibodies that exhibited double masking in Expi293™ cells (Thermo Fisher Scientific, Waltham, MA, Catalog A14635). Order synthetic DNA sequences encoding proteins and signal peptides from Integrated DNA Technologies and clone them into a temporary expression vector containing the CMV promoter. Confirm the endotoxin-free plastid DNA preparation by DNA sequencing before use. Transfect the plastid DNA into Expi293™ cells temporarily using the experimental procedures recommended by the manufacturer. Four days after transfection, the supernatant was harvested and purified using protein A chromatography. For the monomeric population of each expressed protein, use preparative-scale Superdex 200 10/30 GL columns (GE Healthcare Life Sciences, Catalog # 17517501) in 1x PBS pH 7.2 running buffer by particle size exclusion chromatography Method (SEC) purification.

By reducing SDS-PAGE, the purified protein was analyzed. Protein aliquots (5 μg) were denatured in sample buffer with reducing agent for 10 minutes at 75°C and 4-12% NuPAGE ^™ Bis-Tris gel (Thermo Fisher Scientific, Waltham ⁾ in MPOS buffer , MA, Catalog # NP0321) was separated at 15V for 1 hour, stained with InstantBlue ^{™ for} 1 hour, visualized, and then decolorized in water for at least 4 hours. Confirm that all proteins contain two peptides with expected molecular weights (~75KDa and ~25KDa).

Analytical SEC was used to analyze the monomer content of the purified protein. An aliquot of protein (25 μg) was injected onto a Superdex 200 Increase 5/150 GL column (GE Healthcare Life Sciences, Catalog # 28906561) and run at 0.45 ml/min with 1xPBS pH7.2. All proteins are confirmed to contain> 95% monomer content.

The concentration-dependent aggregation of the dual-masked bispecific activatable antibody was evaluated by studying the relationship between monomer content and concentration. An aliquot of purified protein (>95% monomer content) was concentrated using Amicon Ultra 0.5ml centrifugal concentrator (Millipore Sigma, Burlington, MA) at 14,000 rpm in steps (2 minutes each). The percentage of monomer at each concentration was determined by the analytical SEC method described above. As shown in Fig. 1, compared with CI138, CI139 or CI140, the percentage of monomer of CI106 decreases more steeply with concentration. CI139 showed the smallest concentration-dependent aggregation trend, while CI138 and CI140 showed similar concentration-dependent aggregation trends, between CI106 and CI139. The concentration-dependent aggregation trend of these proteins was further tracked by storing the concentrated samples (in PBS) at 4°C for another 2 to 4 days. The SEC results in Table 12 show that the percentages of monomers of CI106 (3% on 2 days), CI138 (4.5% on 4 days), and CI140 (4.3% on 4 days) continued to decrease, but CI139 (0.3% on 4 days) seemed to be stable.

實施例 3 經雙重掩蔽之雙特異性可活化之抗體的 CD3 和 EGFR ELISA 檢定 Each variant was purified by SEC, concentrated with a centrifugal concentrator, and analyzed by SEC-HPLC.

Example 3 CD3 and EGFR ELISA test of bimasked bispecific activatable antibody

The ELISA test is used to confirm and evaluate the binding of complete double-masked bispecific activatable antibodies to CD3 _Ɛ and EGFR ligands. Purified, double-masked, bispecific, activatable anti-body system by combining with recombinant human u-plasminogen activator/urokinase (R&D systems, cat #1310-SE) Incubate for 24 hours to activate, re-purify by protein A chromatography, and re-analyze by SDS-PAGE and SEC.

CD3 _Ɛ ELISA: Human CD3-ε protein (Acro Biosystems, Newark, DE, Catalog CDE-H5223) was coated at 2 μg/ml at 1xPBS pH 7.2 (Thermo Fisher Scientific, Waltham, MA Catalog 20012043) at 4°C overnight On a Nunc MaxiSorp ^™ 96-well plate (Thermo Fisher Scientific, Waltham, MACatalog 439454). Wash the plate with a plate washer at 1x PBS pH 7.2, 0.05% Tween-20, and use at room temperature for 1% BSA, 0.05% Tween 20 in 1x PBS pH 7.2, 200 µL/well block 1 hour. The blocking solution was removed, and a serial dilution of the sample prepared in blocking buffer was added to the blocked plate at room temperature for 1 hour. Before adding the secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG (H+L specificity) diluted 1:25k, Jackson ImmunoResearch Catalog 109-035-088 in blocking buffer), the plate was washed with a plate washer. The signal was developed using 1-Step ^™ TMB-ELISA Substrate solution (Thermo Fisher Scientific, Waltham, MA Catalog 34028), the reaction was stopped with HCl, and the absorbance was read at 450 nm. Figure 2A shows that CI138, CI139, and CI140 behave similarly to CI106, whether in the complete or activated form.

EGFR ELISA: Human EGFR-Fc protein (R & D Biosciences 344-ER ) in 1xPBS pH 7.2 (Thermo Fisher Scientific, Waltham, MA Catalog 20012043) at a concentration of 1μg / ml overnight coating at 4 ^o C in Greiner Bio-One ^TM 96-well plate (Greiner, Monroe, NC, Catalog 655061). Wash the plate with a plate washer at 1x PBS pH 7.2, 0.05% Tween-20, and then use at room temperature for 1% BSA, 0.05% Tween 20 in 1x PBS pH 7.2, blocking at 200 µL/well 1 hour. Samples prepared in blocking buffer were added to blocked plates at room temperature for 1 hour. Before adding the secondary antibody, wash the plate with a plate washer. In the blocking buffer (Jackson ImmunoResearch, West Grove, PA Catalog 209-035-097), a Peroxidase AffiniPure Mouse Anti-Human IgG, F(ab') ₂ fragment diluted 1:5000 was added with a specific secondary antibody. The signal was developed using 1-Step ^™ TMB-ELISA Substrate solution (Thermo Fisher Scientific, Waltham, MA, Catalog 34028), the reaction was stopped with HCl, and the absorbance was read at 450 nm. Figure 2B shows that complete CI138, CI139, and CI140 have similar EGFR binding as CI106. After activation, the EC50 of CI138, CI139, and CI140 looks similar to CI106, but the maximum signal is lower. Example 4 Biological activity of bispecific activatable antibodies with double masking

A cytotoxicity assay was used to determine the biological activity of intact and activated bispecific activatable antibodies. Human PBMC purchased AllCells (Alameda, CA), and HT29-luc2 cells expressing EGFR (Perkin Elmer, Inc., RPMI-1640+glutamax (Sigma, Catalog H3667) supplemented with 5% heat-inactivated human serum) Waltham, MA (formally Caliper Life Sciences, Inc.) was co-cultured at a ratio of 10: 1. The potency of intact and activated CI106, CI138, CI139, and CI140 bispecific activatable antibodies was tested. After 48 hours, The ONE-Glo ^TM Luciferase Assay System (Promega, Madison, WI Catalog E6130) was used to evaluate cytotoxicity. Luminescence was measured on Infinite® M200 Pro (Tecan Trading AG, Switzerland). The percentage of cytotoxicity was calculated and added to the graph graph PRISM. The upper curve fitting analysis. As shown in Figure 3, CI138, CI139, and CI140 are similar to CI106 in the complete form and the activated form. Example 5 The double masked, bispecific, and activatable antibody of the implementation form Induces regression of established HT29-luc2 tumors in mice

In this example, the bispecific activatable antibodies CI106 and CI139 targeting EGFR and CD3ε were analyzed to induce regression or reduce established HT-29-Luc2 xenograft tumors in NSG mice implanted with human T cells The ability to grow.

Human colon cancer cell line HT29-luc2 was obtained from Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.) and cultured according to established procedures. Purified, frozen human PBMC was purchased from Hemacare, Inc., Van Nuys, CA. The NSG ^™ (NOD.Cg- Prkdcscid Il2rg ^tm1Wjl /SzJ) mouse line was obtained from The Jackson Laboratories, Bar Harbor, ME.

實施例 6 經雙重掩蔽、雙特異性、 AA 與 EGFR+ HT-29 細胞和 CD3ε+ Jurkat 細胞的結合 On day 0, the right flank of each mouse was subcutaneously inoculated with 2x10 ⁶ HT29-luc2 cells (in 100 µL RPMI + Glutamax serum-free medium). On day 3, frozen PBMC previously from a single donor were administered (ip) at a ratio of CD3 + T cells to tumor cells of 1:1. When the tumor volume reached 150 mm ³ (approximately day 12), the mice were randomly divided into treatment groups and administered according to Table 13i.v. Tumor volume is measured twice a week. As shown in Figure 5, CI106 and CI139 have equivalent therapeutic effects in this model.

Example 6 Double masking, bispecificity, AA binding to EGFR+ HT-29 cells and CD3ε+ Jurkat cells

To determine whether the described EGFR and CD3ε masking peptides can inhibit binding in double masking, bispecificity, and AA, a binding assay based on flow cytometry was performed.

HT-29-luc2 (Caliper) and Jurkat (E6-1, ATCC, TIB-152) cell lines were cultured in RPMI-1640+glutamax (Life Technologies, Catalog 72400-047), 10% heat-inactivated fetal bovine serum (HI-FBS, Life Technologies, Catalog 10438-026). The following bispecific, activated antibodies CI106 (act-TCB) and double masked, bispecific AA CI106 and CI139 were tested. Two versions of CD3 masking were used, namely CD106 masking in CI106 and CD3 masking in CI139.

HT29-luc2 cells were isolated with Versene™ (Life Technologies, Catalog 15040-066), washed, seeded in 96-well plates at 150,000 cells/well, and then resuspended in 50 µL of primary antibody. Count and seed Jurkat cells as described for HT29-luc2 cells. The titer of the primary antibody starts at the concentration shown in Figure 6, and is then serially diluted 3-fold in FACS staining buffer + 2% FBS (BD Pharmingen, Catalog 554656). The cells were incubated at 4°C with shaking for about 1 hour, collected and washed with 2x200 µL of FACS staining buffer. The cells were resuspended in 50 µL Alexa Fluor 488 conjugated anti-human IgG Fc (10 µg/ml, Jackson ImmunoResearch), and incubated at 4°C with shaking for about 1 hour. The cells were harvested, washed and resuspended in a final volume of 200 µL of FACS staining buffer containing 2.5 µg/ml 7-AAD (BD Biosciences, Catalog 559925). Cells stained with secondary antibody alone were used as negative controls. Data was acquired on an Attune NxT flow cytometer, and FlowJo ^® V10 (Treestar) was used to calculate the median fluorescence intensity (MFI) of viable cells. Using curve fitting analysis, the MFI data for background subtraction was plotted in GraphPad Prism.

Figure 6 depicts the reduced binding of the complete activatable bispecific antibodies CI106 and CI139 to EGFR and CD3 relative to the activated bispecific antibody, as shown by the right shift of the binding curve. Example 7 Biological activity of bispecific activatable antibodies with double masking

A cytotoxicity test is used to test the biological activity of intact and activated bispecific activatable antibodies. Human PBMC was purchased from Stemcell Technologies (Vancouver, Canada), and compared with EGFR-expressing cancer cell line HT29-luc2 (Perkin Elmer) in RPMI-1640+glutamax (Sigma, Catalog H3667) supplemented with 5% heat-inactivated human serum , Inc., Waltham, MA (formally Caliper Life Sciences, Inc.), NCI-N87 (ATCC), OE33 (ATCC), SKBR3 (ATCC), or SKOV3 (ATCC) at a ratio of 10:1, 6:1, respectively 8:1, 6:1, or 6:1 co-culture. Test the potency of activated CI106 (act-TCB) and complete CI106 and CI139 bispecific activatable antibodies. After 48 hours, use ONE-Glo ^TM Luciferase Assay System (Promega, Madison, WI Catalog E6130) to evaluate cytotoxicity. Luminescence is measured on Infinite® M200 Pro (Tecan Trading AG, Switzerland). Calculate the percentage of cytotoxicity, and plot on GraphPad PRISM plus curve fitting analysis.

實施例 8 實施態樣的經雙重掩蔽、雙特異性、可活化之抗體誘導小鼠中已建立的 HCT116 腫瘤的消退 As shown in FIGS. 7A-C and Table 14, intact bispecific activatable antibodies have varying dose response curves relative to activated bispecific antibodies, and CI139 activity is similar to CI106 in these assays.

Example 8 Implementation of double masked, bispecific, activatable antibodies induces regression of established HCT116 tumors in mice

In this example, the ability of bispecific activatable antibodies CI106 and CI139 targeting EGFR and CD3ε to induce regression or reduce the growth of established HCT116 xenograft tumors in NSG mice implanted with human T cells was analyzed.

Human colon cancer cell line HCT116 was obtained from ATCC and cultured in RPMI + Glutamax + 10% FBS according to established procedures. The tumor pattern was performed as described in Example 5. As shown in Figure 8, CI106 and CI139 have equivalent therapeutic effects in this model. Example 9 Pharmacokinetics of double-masked BAA in crab-eating macaques

In this example, in cynomolgus macaques, the dual-masked, bispecific antibodies CI106 and CI139 were used at 3 mg/kg on day 1, 6 mg/kg on day 8, and on day 15 Give at 6 mg/kg. Plasma samples were collected 30 minutes, 4h, 24h, 96h, and 168h after the dose on Days 1 and 8 and 30 minutes, 4h, 24h, and 72 hr after the dose on Day 15. The plasma concentration was measured by ELISA using a universal Fc capture and detection method. Plasma concentration values were interpolated from the standard curve and plotted using GraphPad PRISM. Figure 9 depicts the pharmacokinetics of double-masked molecules CI106 and CI139 in cynomolgus macaques.

Although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended patent application. Other aspects, advantages, and modifications are within the scope of the following.

Figure 1 confirms the concentration-dependent aggregation of the double-masked bispecific activatable antibody of Table 11, which was evaluated by studying the relationship between monomer content and concentration.

Figure 2A demonstrates the combination of intact (unactivated) and activated BAA CI138, CI139, and CI140 compared to CI106 as assessed by CD3ε-based ELISA.

Figure 2B demonstrates the binding of intact and activated BAA CI138, CI139, and CI140 compared to CI106 as assessed by EGFR-based ELISA.

Figure 3 demonstrates the biological activity of intact and activated BAA CI138, CI139, and CI140 as compared to CI106 as assessed by the cytotoxicity assay.

Figure 4 depicts an exemplary BAA provided herein.

Figure 5 plots the tumor volume against the number of days after the initial therapeutic dose, demonstrating the dose-dependent effect of CI106 and CI139 on the growth of HT29-luc2 xenograft tumors through double masking, bispecific, and activatable antibodies. The most effective dose tested was 3.0 mg/kg, which caused tumor regression.

Figure 6 confirms the binding of CI106 and CI139 to HT29 and Jurkat cells compared to activated bispecific antibodies via a binding assay based on flow cytometry.

Figure 7 demonstrates the biological activity of intact and activated BAA CI139 compared to CI106 as assessed by cytotoxicity assays in different cell lines.

Figure 8 plots the tumor volume against the number of days after the initial therapeutic dose, confirming the dose-dependent effect of CI106 and CI139 on the growth of HCT116 xenograft tumors through double masking, bispecific, and activatable antibodies.

Figure 9 confirms the pharmacokinetics of double-masked molecules CI106 and CI139 in cynomolgus macaques.

Claims (69)

A bispecific activatable antibody (BAA), wherein the BAA specifically binds to two targets when activated, and wherein the BAA contains the following structure when not activated: a) IgG antibody (AB1) that specifically binds to the first target, where AB1 contains: i. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. Two first pre-domains, each of which contains a first masking portion (MM1) connected to the first cleavable portion (CM1) from the N-terminal to the C-terminal direction, wherein the carboxyl group of each first pre-domain The end is connected to the amine end of each light chain of AB1, where -The MM1 reduces the binding of the AB1 to its target; and -This CM1 peptide is used as a substrate for the first protease, b) Two scFv (AB2), each of which specifically binds CD3ε, where each AB2 contains: i. The light chain variation region (VL) connected to the heavy chain variation region (VH), wherein the carboxyl terminal of each AB2 is connected to the amine terminal of each of the AB1 heavy chains, of which -The VL contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH contains the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and -The VL is connected to the VH by a linker L3, the linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and ii. Two second pre-domains, each of which contains a second masking portion (MM2) connected to the second cleavable portion (CM2) in the direction from N-terminal to C-terminal, wherein the carboxyl group of each second pre-domain The end is connected to the amine end of each AB2, where -The MM2 reduces the combination of AB2 and CD3ε; -The MM2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and -The CM2 peptide is used as a substrate for the second protease. A BAA according to claim 1, wherein the VL is connected to the VH through a linker comprising the amino acid sequence SEQ ID NO: 98. A BAA according to claim 1, wherein the VL is connected to the VH through a linker comprising the amino acid sequence SEQ ID NO: 108. The BAA according to any one of claims 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 12. The BAA of any one of claims 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 105. The BAA of any one of claims 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 106. The BAA according to any one of claims 1 to 3, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 107. The BAA of any one of claims 1 to 7, wherein the AB1 binds to a tumor target. The BAA of any one of claims 1 to 8, wherein the AB1 binds EGFR. A BAA according to any one of claims 1 to 9, wherein the MM1 comprises an amino acid sequence selected from the group consisting of the sequences presented in Table 7 or Table 8. The BAA of any one of claims 1 to 10, wherein the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 85. The BAA of any one of claims 1 to 11, wherein the MM1 comprises the amino acid sequence SEQ ID NO: 78. The BAA according to any one of claims 1 to 11, wherein the MM1 comprises the amino acid sequence SEQ ID NO: 85. A BAA according to any one of claims 0 to 13, wherein the CM1 or CM2 comprises the amino acid sequence of any of the CMs listed in Table 4 or Table 4-1. A BAA according to any one of claims 0 to 14, wherein the CM1 or CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17. A BAA according to any one of claims 0 to 15, wherein the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16. The BAA according to any one of claims 0 to 15, wherein the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17. A BAA according to any one of claims 0 to 17, wherein the AB1 includes an Fc region, which includes at least one of amino acid positions L234, L235, N297, and P331 (numbered by the EU index shown in Kabat) Amino acid substitution makes the BAA have reduced effector function. As in the BAA of claim 18, wherein the AB1 includes amino acid substitutions in at least two of amino acid positions L234, L235, and P331. A BAA according to any one of claims 18 to 19, wherein the AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P331. The BAA according to any one of claims 18 to 20, wherein the AB1 includes amino acid substitutions of L234F, L235E, and P331S. A BAA according to any one of claims 0 to 21, wherein the AB1 comprises an Fc region, which comprises an amino acid substitution in N297. A BAA according to any one of claims 18 to 22, wherein the AB1 includes amino acid substitutions of L234F, L235E, P331S, and N297Q. The BAA according to any one of claims 18 to 23, wherein the heavy chain of AB1 comprises the amino acid sequence shown in Table 4-2 selected from the group consisting of: SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122. The BAA according to any one of claims 18 to 23, wherein the heavy chain of AB1 comprises the amino acid sequence shown in Table 6 selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76. A BAA according to any one of claims 0 to 0 and 0 to 0, wherein the first target is selected from the group consisting of the targets presented in Table 9. A BAA according to any one of claims 0 to 0, wherein the first and second front domains each include a linker L1 according to the formula (MM)-L1-(CM)-L2 in the N-terminal to C-terminal direction And linker L2. A BAA (CI138), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 155 or SEQ ID NO: 124, and the light chain amino group shown in SEQ ID NO: 132 or SEQ ID NO: 140 Acid sequence. The BAA (CI139), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 157 or SEQ ID NO: 126, and the light chain amino group shown in SEQ ID NO: 132 or SEQ ID NO: 140 Acid sequence. The BAA (CI158), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 161 or SEQ ID NO: 128, and the light chain amino group shown in SEQ ID NO: 132 or SEQ ID NO: 140 Acid sequence. The BAA (CI140), wherein the BAA comprises the heavy chain amino acid sequence shown in SEQ ID NO: 159 or SEQ ID NO: 25, and the light chain amino group shown in SEQ ID NO: 132 or SEQ ID NO: 140 Acid sequence. An activatable antibody (AA), wherein the AA specifically binds to the target when activated, and wherein the AA contains the following structure when not activated: a) at least one scFv, which comprises a light chain variant region (VL) connected to a heavy chain variant region (VH), wherein the VL is connected to the VH by a linker L3, the linker comprises a member selected from the group consisting of SEQ ID NO: The amino acid sequence of the group consisting of 98 and SEQ ID NO: 108; and b) Prodomain, which contains: i. A masking moiety (MM) coupled to the scFv, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the scFV to its target; and ii. A truncable portion (CM) coupled to the scFv, wherein the CM is a substrate polypeptide used as a protease. As in AA of claim 0, the target of the scFv is CD3ε. An activatable antibody (AA), wherein the AA specifically binds to the target when activated, and wherein the AA contains the following structure when not activated: a) antibodies or antigen-binding fragments (AB) that specifically bind to CD3ε; and b) Prodomain, which contains: i. A masking part (MM) coupled to the AB, wherein when the AA is in an uncut state, the MM reduces or inhibits the binding of the AB and CD3ε, wherein the MM includes the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and ii. A cleavable portion (CM) coupled to the AB, wherein the CM is a substrate polypeptide used as a protease. AA according to any one of claims 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13-17. AA according to any one of claims 32 to 34, wherein the CM comprises a substrate which can be cut by serine protease or MMP. AA according to any one of claims 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-56. The AA of any one of claims 32 to 34, wherein the protease is MMP. AA according to any one of claims 32 to 34, wherein the protease is serine protease. AA according to any one of claims 32 to 39, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, and the VH comprises SEQ ID NO: 2 or SEQ ID NO: 3 shows the amino acid sequence. The AA according to any one of claims 32 to 40, wherein according to the formula (MM)-L1-(CM)-L2 in the N-terminal to C-terminal direction, the pre-domain includes a linker L1 and a linker L2. AA or BAA according to any one of claims 0 to 0, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, F(ab') ₂ fragment, scFv, scAb, dAb, single domain Heavy chain antibodies, and single domain light chain antibodies. The AA or BAA of any one of claims 0 to 0, wherein the anti-systemic rodent antibody, chimeric antibody, humanized antibody, or fully human monoclonal antibody. AA as in any one of claims 0 to 0, where the AA is part of the BAA. A pharmaceutical composition comprising AA or BAA according to any one of claims 0 to 0 and optionally a carrier. The pharmaceutical composition according to claim 45 contains other agents. The pharmaceutical composition according to claim 46, wherein the other agent is a therapeutic agent. An isolated nucleic acid molecule that encodes AA or BAA according to any one of claims 0 to 0. A vector comprising the isolated nucleic acid molecule according to claim 0. A vector comprising the nucleic acid sequence of pEF1049. A vector comprising the nucleic acid sequence of pEF1050. A vector comprising the nucleic acid sequence of pEF1107. A vector comprising the nucleic acid sequence of pEF1052. A cell comprising the vector according to any one of claims 49 to 53. A cell comprising pEF1049 and pLW246. A cell comprising pEF1050 and pLW246. A cell comprising pEF1107 and pLW246. A cell comprising pEF1052 and pLW246. A method of manufacturing an AA or BAA according to any one of claims 0 to 44 by culturing a cell under conditions that result in expression of AA or BAA, wherein the cell contains the nucleic acid molecule according to claim 48 or according to claim 49 to The vector of any one of 53, or the cell of any one of claims 54 to 58. A method of treating a disease or disease, reducing symptoms, or delaying progression, which comprises treating a therapeutically effective amount of AA or BAA as in any one of claims 0 to 44, or as in any one of claims 45 to 47 The pharmaceutical composition is administered to individuals who need it. The method of claim 60, wherein the condition or disease comprises disease cells expressing EGFR. The method of any one of claims 60 or 61, wherein the condition or disease is cancer. The method of claim 62, wherein the cancer is bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, Ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, squamous cell carcinoma, or skin cancer. A method of inhibiting angiogenesis in an individual, comprising administering a therapeutically effective amount of AA or BAA as in any one of claims 0 to 44, or a pharmaceutical composition as in any one of claims 45 to 47 Individual. The method according to any one of claims 60 to 64, wherein the method comprises administration of other agents. The method of claim 65, wherein the other agent is a therapeutic agent. A method for reducing damage to healthy tissue caused by antibody binding to its target on healthy tissue and also on diseased tissue, the method comprising administering AA, BAA or a pharmaceutical composition containing AA or BAA to another An individual, wherein the AA or BAA is the AA or BAA of any one of claims 1 to 0. A method for improving the tolerance of antibody treatment, comprising administering AA, BAA or a pharmaceutical composition containing AA or BAA to an individual in need thereof, wherein the AA or BAA is as in any one of claims 1 to 0 AA or BAA. A method for recruiting T cells to tumor tissues, comprising administering AA or BAA or a pharmaceutical composition containing AA or BAA to an individual in need thereof, wherein the AA or BAA is as in any one of claims 1 to 0 Of AA or BAA.

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